cell bio exam #1

SAM (sorting and assembly machinery)

helps mitochondrian proteins to fold properly in the outer membrane.

ATP-driven pumps

hydrolyze ATP to ADP and phosphate and use the energy released to pump ions or other solutes across a membrane

TIM22 (translocator of the inner mitochondrial membrane)

mediates the insertion of a subclass of inner membrane proteins, including the transporter that moves ADP, ATP, and phosphate in and out of mitochondria.

OXA complex

mediates the insertion of those inner membrane proteins that are synthesized within mitochondria and some imported inner membrane proteins.

bright field light microscope

most widely used, specimen is darker than surrounding field

scramblases

nonselectively equilibrate phospholipids between the two leaflets of the lipid bilayer. Thus, the different types of phospholipids are thought to be equally distributed between the two leaflets of the ER membrane.

uniporters

passively mediate the movement of a single solute from one side of the membrane to the other

functions of plasma membrane

physical isolation, regulation of exchange of information with the environment, import and export of molecules, structural support, capacity for movement and expansion

TOM (translocator of the outer mitochondrial membrane)

required for the import of all nucleus-encoded mitochondrial proteins.

how do cells replicate their hereditary information?

templated polymerization

numerical aperture

the higher the numerical aperture, the greater the resolution and the brighter the image (brightness is important in fluorescence microscopy)

resolution

the resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens

swinging bucket rotor

the sample tubes are placed in metal tubes on hinges that allow the tubes to swing outward when the rotor spins. Sample tubes are therefore horizontal during spinning, and samples are sedimented toward the bottom, not the sides, of the tube, providing better separation of differently sized components.

autocatalytic fluorescent protein chromophore formation

three residues, Ser65, Tyr66, and Gly67, can form into a mature GFp chromophore (which allows for green fluorescence)

antiporters

transfer of one solute depends on the simultaneous transfer of a second solute in the opposite direction

symporters

transfer of one solute depends on the simultaneous transfer of a second solute in the same direction

TIM23 (translocator of the inner mitochondrial membrane)

transports some soluble proteins into the matrix space and helps to insert transmembrane proteins into the inner membrane.

hydrophobic forces

water forces hydrophobic groups together because doing so minimizes their disruptive effects on the hydrogen-bonded water network. hydrophobic groups held together in this way are sometimes said to be held together by "hydrophobic bonds" even though the apparent attraction is actually caused by a repulsion from the water.

variety of cell size and shape

•Cells vary enormously in size and shape •Cells arise only from preexisting cells. •Every cell has genetic information whose expression enables it to produce all its components.

makeup of prokaryotic cells

•The plasma membrane of a prokaryote surrounds a single compartment. •The entire compartment has the same aqueous environment. •Genetic material occupies a compact area within the cell. •The plasma membrane is surrounded by a cell wall, whose rigid structure gives physical protection against the environment. •Bacteria and archaea are both prokaryotes but differ in some structural features.

the role of energy in protein import into the mitochondrial matrix space

(1) Bound cytosolic Hsp70 is released from the protein, depending on ATP hydrolysis. After initial insertion of the signal sequence and of adjacent portions of the polypeptide chain into the TOM complex, the signal sequence interacts with a TIM complex. (2) The signal sequence (positively charged) is then translocated into the matrix space in a process that requires a membrane potential across the inner membrane. (electrochemical gradient of H+) (3) Mitochondrial Hsp70, which is part of an import ATPase complex, binds to regions of the polypeptide chain as they become exposed in the matrix space, pulling the protein through the translocation channel (acts as a motor).

role of phospholipid translocators in lipid bilayer synthesis

(A) Because new lipid molecules are added only to the cytosolic half of the ER membrane bilayer and lipid molecules do not flip spontaneously from one monolayer to the other, a transmembrane phospholipid translocator (called a scramblase) is required to transfer lipid molecules from the cytosolic half tothe lumenal half so that the membrane grows as a bilayer. The scramblase is not specific for particular phospholipid head groups and therefore equilibrates the different phospholipids between the two monolayers. (B) Fueled by ATP hydrolysis, a head-group-specific flippase in the plasma membrane actively flips phosphatidylserine and phosphatidylethanolamine directionally from the extracellular to the cytosolic leaflet, creating the characteristically asymmetric lipid bilayer of the plasma membrane of animal cells

three ways in which protein translocation can be driven through structurally similar translocators

(A) Co-translational translocation. The ribosome is brought to the membrane by the SRP and SRP receptor and then engages with the Sec61 protein translocator. The growing polypeptide chain is threaded across the membrane as it is made. No additional energy is needed, as the only path available to the growing chain is to cross the membrane. (B) Post-translational translocation in eukaryotic cells requires an additional complex composed of Sec62, Sec63, Sec71, and Sec72 proteins, which is attached to the Sec61 translocator and deposits BiP molecules onto the translocating chain as it emerges from the translocator in the lumen of the ER. ATP-driven cycles of BiP binding and release pull the protein into the lumen, a mechanism that closely resembles the mechanism of mitochondrial import in Figure 12-23. (C) Post-translational translocation in bacteria. The completed polypeptide chain is fed from the cytosolic side into the bacterial homolog of the Sec61 complex (called the SecY complex in bacteria) in the plasma membrane by the SecA ATPase. ATP hydrolysis-driven conformational changes drive a pistonlike motion in SecA, each cycle pushing about 20 amino acids of the protein chain through the pore of the translocator. The Sec pathway used for protein translocation across the thylakoid membrane in chloroplasts uses a similar mechanism

signal-recognition particle (SRP)

- A mammalian SRP is a rodlike complex containing six protein subunits and one RNA molecule. The SRP RNA forms the backbone that links the protein domain of the SRP containing the signal sequence binding pocket to the domain responsible for pausing translation. - Cycles between the ER mem- brane and the cytosol and binds to the signal sequence - The 3D outline of the SRP bound to a ribosome was determined by cryo-EM. SRP binds to the large ribosomal subunit so that its signal-sequence-binding pocket is positioned near the growing polypeptide chain exit site, and its translational pause domain is positioned at the interface between the ribosomal subunits, where it interferes with elongation factor binding.

electron microscope (EM) tomography

- A method that combines a set of different EM views of a single object in three dimensions and take an average, thus the noise of individual images can be largely eliminated, yielding a clear view of the molecular structure. - Different views of a single object can be combined to give a 3D reconstruction. - Similar computational methods are used in medical CT scans. - Starting with thick plastic sections of embedded material, three-dimensional reconstructions, or tomograms, are used extensively to describe the detailed anatomy of specific regions of the cell, such as the Golgi apparatus or the cytoskeleton. - Increasingly, microscopists are also applying EM tomography to unstained frozen, hydrated sections, and even to rapidly frozen whole cells or organelles

column chromatography

- A mixture of proteins in solution is passed through a column containing a porous solid matrix. - Different proteins are retarded to different extents by their interaction with the matrix, and they can be collected separately as they flow out of the bottom of the column

ER signal sequence

- A short amino acid sequence that marks a polypeptide for transport to the endoplasmic reticulum, where synthesis of the polypeptide chain is completed and the signal sequence removed. - First signal sequences discovered in secreted proteins that are translocated across the ER membrane. - When the ER signal sequence emerges from the ribosome, it directs the ribosome to a translocator on the ER membrane (a pore in the membrane through which the polypeptide is translocated). A signal peptidase is closely associated with the translocator and clips off the signal sequence during translocation, and the mature protein is released into the lumen of the ER immediately after synthesis. The translocator is closed until the ribosome has bound, so that the permeability barrier of the ER membrane is maintained at all times.

bacteriorhodopsin

- A single species of protein molecule that functions as a light-activated H+ pump that transfers H+ out of the archaeal cell. - Each bacteriorhodopsin molecule is folded into seven closely packed trans- membrane α helices and contains a single light-absorbing group, or chromophore (in this case, retinal), which gives the protein its purple color. - Retinal is covalently linked to a lysine side chain of the bacteriorhodopsin protein. When activated by a single photon of light, the excited chromophore changes its shape and causes a series of small conformational changes in the protein, resulting in the transfer of one H+ from the inside to the outside of the cell. - In bright light, each bacteriorhodopsin molecule can pump several hundred protons per second. The light-driven proton transfer establishes an H+ gradient across the plasma membrane, which in turn drives the production of ATP by a second protein in the cell's plasma membrane. The energy stored in the H+ gradient also drives other energy-requiring processes in the cell. Thus, bacteriorhodopsin converts solar energy into a H+ gradient, which provides energy to the archaeal cell.

ABC transporters

- ABC transporters constitute the largest family of membrane transport proteins and are of great clinical importance. - Differ structurally from P-type ATPases and primarily pump small molecules across cell membranes. - Consist of multiple domains, typically, two hydrophobic domains, each built of six membrane-spanning segments that form the translocation pathway and provide substrate specificity, and two ATPase domains (also called ATP-binding cassettes) protruding into the cytosol. - Without ATP bound, the transporter exposes a substrate-binding site to either the extracellular space (in procaryotes) or the intracellular space (in eucaryotes or procaryotes). ATP binding induces a conformational change that exposes the substrate-binding pocket to the opposite face; ATP hydrolysis followed by ADP dissociation returns the transporter to its original conformation. - Most individual ABC transporters are unidirectional. Both importing and exporting ABC transporters are found in bacteria, but in eucaryotes almost all ABC transporters export substances from the cytosol either to the extracellular space or to a membrane-bound intracellular compartment such as the ER or the mitochondria.

passive transport

- All channels and many transporters allow solutes to cross the membrane only passively - for a single uncharged molecule, the concentration gradient drives passive transport and determines its direction - If the solute carries a net charge, how- ever, both its concentration gradient and the electrical potential difference across the membrane, the membrane potential, influence its transport. - The concentration gradient and the electrical gradient combine to form a net driving force, the electrochemical gradient, for each charged solute - Passive transport down an electrochemical gradient occurs spontaneously, either by simple diffusion through the lipid bilayer or by facilitated diffusion through channels and passive transporters

endoplasmic reticulum

- All eukaryotic cells have an ER. - Its membrane typically constitutes more than half of the total membrane of an average animal cell. - The ER is organized into a netlike labyrinth of branching tubules and flattened sacs that extends throughout the cytosol. - It is structurally and functionally diverse; while the various functions of the ER are essential to every cell, their relative importance varies greatly between individual cell types. - To meet different functional demands, distinct regions of the ER become highly specialized (causing dramatic changes in ER structure)

action potential

- An electrical stimulus that exceeds a certain threshold strength, triggering an explosion of electrical activity that propagates rapidly along the neuron's plasma membrane and is sustained by automatic amplification all along the way. - Direct consequence of the properties of voltage-gated cation channels. - Triggered by a depolarization of the plasma membrane—that is, by a shift in the membrane potential to a less negative value inside. - In nerve and skeletal muscle cells, a stimulus that causes sufficient depolarization promptly opens the voltage-gated Na+ channels, allowing a small amount of Na+ to enter the cell down its electrochemical gradient. The influx of positive charge depolarizes the membrane further, thereby opening more Na+ channels, which admit more Na+ ions, causing still further depolarization. This self-amplification process (an example of positive feedback) continues until, within a fraction of a millisecond, the electrical potential in the local region of membrane has shifted from its resting value of about -70 mV (in squid giant axon; about -40 mV in human) to almost as far as the Na+ equilibrium potential of about +50 mV. At this point, when the net electrochemical driving force for the flow of Na+ is almost zero, the cell would come to a new resting state, with all of its Na+ channels permanently open, if the open conformation of the channel were stable. Two mechanisms act in concert to save the cell from such a permanent electrical spasm: the Na+ channels automatically inactivate and voltage-gated K+ channels open to restore the membrane potential to its initial negative value.

optical system of a fluorescence microscope

- An orbital electron of a fluorochrome molecule can be raised to an excited state following the absorption of a photon. - Fluorescence occurs when the electron returns to its ground state and emits a photon of light at a longer wavelength. - (B) shows the filter set for detection of the fluorescent molecule fluorescein (green color).

Protein import into mitochondria

- As a first step in the import process, the import receptors of the TOM complex bind the signal sequence of the mitochondrial precursor protein. - The interacting proteins are then stripped off, and the unfolded polypeptide chain is fed—signal sequence first—into the translocation channel. - The protein is then translocated through the TIM23 complex so that it transiently spans both mitochondrial membranes. - The signal sequence is cleaved off by a signal peptidase in the matrix space to form the mature protein. The free signal sequence is then rapidly degraded. - Although the TOM and TIM complexes usually work together to transport precursor proteins across both membranes at the same time, they can work independently.

nuclear transport of baculovirus

- Baculoviruses are large, rod-shaped (30-60 x 250-300 nm), enveloped viruses with a DNA genome that requires the nuclear machinery for viral replication - Electron micrographs of NPC cross-sections from Xenopus oocytes that have been microinjected with baculovirus AcMNPV capsid and incubated at room temperature for 3.5 h. Capsids of 250-300 nm in length are seen traversing the NPCs. Capsids appear fully intact in its native conformation while crossing the NPC.

how can transport through NPCs be regulated?

- By controlling access to the transport machinery - Cells do this by regulating nuclear localization and export signals, turning them on or off, often by phosphorylation of amino acids close to the signal sequences. - Controlling nuclear import during T-cell activation: The nuclear factor of activated T cells (NF-AT) is a gene regulatory protein that, in the resting T cell, is found in the cytosol in a phosphorylated state. When T cells are activated by foreign antigen, the intracellular Ca2+ concentration increases. In high Ca2+, the protein phosphatase calcineurin binds to NF-AT and dephosphorylates it. The dephosphorylation exposes nuclear import signals and blocks a nuclear export signal. The complex of NF-AT and calcineurin is then imported into the nucleus, where NF-AT activates the transcription of numerous genes required for T cell activation. The response shuts off when Ca2+ levels decrease, releasing NF-AT from calcineurin. Rephosphorylation of NF-AT inactivates the nuclear import signals and re-exposes the nuclear export signal, causing NF-AT to relocate to the cytosol. - Some of the most potent immunosuppressive drugs, including cyclosporin A and FK506, inhibit the ability of calcineurin to dephosphorylate NF-AT and thereby block the nuclear accumulation of NF-AT and T cell activation.

evolution of internal membranes

- Clear homologs of actin, tubulin, histones, and the nuclear DNA replication system are found in archaea, but not in bacteria. Thus, it is now thought that the first eukaryotic cells arose when an ancient anaerobic archaeon joined forces with an aerobic bacterium. - The nuclear envelope may have originated from an invagination of the plasma membrane of this ancient archaeon—an invagination that protected its chromosome while still allowing access of the DNA to the cytosol (as required for DNA to direct protein synthesis). This envelope may have later pinched off completely from the plasma membrane, so as to produce a separate nuclear compartment surrounded by a double membrane. Because this double membrane is penetrated by nuclear pore complexes, the nuclear compartment is topologically equivalent to the cytosol. in contrast, the lumen of the ER is continuous with the space between the inner and outer nuclear membranes, and it is topologically equivalent to the extracellular space.

topologically equivalent compartments

- Compartments are said to be topologically equivalent if they can communicate with one another, in the sense that molecules can get from one to the other without having to cross a membrane. Topologically equivalent spaces are shown in orange. In principle, cycles of membrane budding and fusion permit the lumen of any of these organelles to communicate with any other and with the cell exterior by means of transport vesicles. Blue arrows indicate the extensive outbound and inbound vesicular traffic. - Some organelles, most notably mitochondria and (in plant cells) plastids, do not take part in this communication and are isolated from the traffic between organelles shown here.

three ways in which membrane-bending proteins shape membranes

- Cytoskeletal dynamics and membrane-bending-protein forces often work together to deform (or bend) lipid bilayers. 1. A hydrophobic region of the protein can insert as a wedge into one monolayer to pry lipid head groups apart. Such regions can either be amphiphilic helices as shown or hydrophobic hairpins. 2. The curved surface of the protein can bind to lipid head groups and deform the membrane or stabilize its curvature. 3. A protein can bind to and cluster lipids that have large head groups and thereby bend the membrane. * In contrast to (D), phospholipases that remove lipid head groups produce inversely shaped lipid molecules that induce negative curvature.

applications of fluorescence microscopy

- Determine the location of a particular molecule/cellular structure using fixed samples or live cells; - Monitor changes in the concentration and location of specific molecules; - Follow protein dynamics in a living cell - Introduce a fluorescent gene marker and track its expression during development.

ER assembles most lipid bilayers

- ER membrane is the site of synthesis of nearly all of the cell's major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. - Each step is catalyzed by enzymes in the ER membrane, which have their active sites facing the cytosol, where all of the required metabolites are found. - Thus, phospholipid synthesis occurs exclusively in the cytosolic leaflet of the ER membrane.

function of transporters in passive transport

- Each type of transporter has one or more specific binding sites for its solute (substrate). - It transfers the solute across the lipid bilayer by undergoing reversible conformational changes that alternately expose the solute-binding site first on one side of the membrane and then on the other—but never on both sides at the same time. - There are three conformational states: in the outward-open state, the binding sites for solute are exposed on the outside of the lipid bilayer; in the occluded state, the same sites are not accessible from either side; and in the inward-open state, the sites are exposed on the inside of the bilayer. - The transition between the states occur randomly. - They are completely reversible and do not depend on whether the solute binding site is occupied. - Therefore, if the solute concentration is higher on the outside of the bilayer, more solute binds to the transporter in the outward-open conformation than in the inward-open conformation, and there is a net transport of solute down its concentration gradient

immunogold electron microscopy

- Electron microscopy technique in which cellular structures or molecules of interest are labeled with antibodies tagged with electron-dense gold particles. - The usual procedure is to incubate a thin section first with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electron-dense and can be seen as a black dot in the electron microscope. Different antibodies can be conjugated to different sized gold particles so multiple proteins can be localized in a single sample.

membrane-bound ribosomes

- Engaged in the synthesis of proteins that are being concurrently translocated into the ER - Attached to the cytosolic side of the ER membrane - membrane-bound and free ribosomes are structurally and functionally identical.

glycolipids on plasma membranes

- Found exclusively in the monolayer facing away from the cytosol. - In animal cells, they are made from sphingosine, just like sphingomyelin. - These intriguing molecules tend to self-associate, partly through hydrogen bonds between their sugars and partly through van der Waals forces between their long and straight hydrocarbon chains, which causes them to partition preferentially into lipid raft phases.

Na+-K+ pump

- Found in the plasma membrane of virtually all animal cells - Belongs to the family of P-type ATPases - Operates as an ATP-driven antiporter. - The Na+-K+ pump actively pumps Na+ out of and K+ into a cell against their electrochemical gradients. For every molecule of ATP hydrolyzed the pump, three Na+ are pumped out and two K+ are pumped in.

scanning electron microscope

- In a SEM, the specimen is scanned by a beam of electrons brought to a focus on the specimen by the electromagnetic coils that act as lenses. The detector measures the quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen and controls the intensity of successive points in an image built up on a video screen. The SEM creates striking images of 3D objects with great depth of focus and a resolution between 3 nm and 20 nm depending on the instrument. - The SEM technique provides great depth of field; moreover, since the amount of electron scattering depends on the angle of the surface relative to the beam, the image has highlights and shadows that give it a three-dimensional appearance

Cells can confine proteins and lipids to specific domains within a membrane

- In an epithelial cell, protein A and protein B can diffuse laterally in their own domains but are prevented from entering the other domain, at least partly by the specialized cell junction called a tight junction. - Lipid molecules in the outer (noncytosolic) monolayer of the plasma membrane are likewise unable to diffuse between the two domains; lipids in the inner (cytosolic) monolayer, however, are able to do so (not shown). The basal lamina is a thin mat of extracellular matrix that separates epithelial sheets from other tissues.

insertion of the multipass membrane protein Rhodopsin into the ER membrane

- In more complex multipass proteins, in which many hydro- phobic α helices span the bilayer, a second start-transfer sequence reinitiates translocation further down the polypeptide chain until the next stop-transfer sequence causes polypeptide release, and so on for subsequent start-transfer and stop-transfer sequences - A hydrophobicity (also called hydropathy) plot identifies seven short hydrophobic regions in rhodopsin. - The hydrophobic region nearest the N-terminus serves as a start-transfer sequence that causes the preceding N-terminal portion of the protein to pass across the ER membrane. Subsequent hydrophobic sequences function in alternation as start-transfer and stop-transfer sequences. - The final integrated rhodopsin has its N-terminus located in the ER lumen and its C-terminus located in the cytosol. The blue hexagons represent covalently attached oligosaccharides. Arrows indicate the paired start and stop signals inserted into the translocator.

Ran

- In order to fuel the import of nuclear proteins, energy is harnessed from that stored in the form of concentration gradients in GTP-bound form of the monomeric GTPase Ran, which is required for both nuclear import and export. - Ran is a molecular switch that can exist in two conforma- tional states, depending on whether GDP or GTP is bound. - wo Ran-specific regulatory proteins trigger the conversion between the two states: a cytosolic GTPase-activating protein (GAP) triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) promotes the exchange of GDP for GTP and thus converts Ran-GDP to Ran-GTP. - Because Ran-GAP is located in the cytosol and Ran-GEF is located in the nucleus where it is anchored to chromatin, the cytosol contains mainly Ran-GDP, and the nucleus contains mainly Ran-GTP.

structure of aquaporin

- In the membrane, aquaporins form tetramers, with each monomer (A & B) containing an aqueous pore in its center. - Each individual aquaporin channel passes about 109 water molecules per second. - One face of the pore is lined with hydrophilic amino acids, which provide transient hydrogen bonds to water molecules via carbonyl oxygens; these bonds help line up the transiting water molecules in a single row and orient them as they traverse the pore. - The central two Asn bind to the oxygen atom of the central water molecule, imposing a bipolarity on the entire column of water molecules

velocity sedimentation vs equilibrium sedimentation

- In velocity sedimentation, subcellular components sediment at different speeds according to their size and shape when layered over a solution containing sucrose. To stabilize the sedimenting bands against convective mixing caused by small differences in temperature or solute concentration, the tube contains a continuous shallow gradient of sucrose, which increases in concentration toward the bottom of the tube (typically from 5% to 20% sucrose). After centrifugation, the different components can be collected individually, most simply by puncturing the plastic centrifuge tube with a needle and collecting drops from the bottom, as illustrated here. - In equilibrium sedimentation, subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings. Although a sucrose gradient is shown here, denser gradients, which are especially useful for protein and nucleic acid separation, can be formed from cesium chloride. The final bands, at equilibrium, can be collected as in (A).

glucose transport fueled by a Na+ gradient

- Intestinal and kidney epithelial cells contain a variety of symporters that are driven by the Na+ gradient across the plasma membrane. Each Na+-driven symporter is specific for importing a small group of related sugars or amino acids into the cell. Because the Na+ tends to move into the cell down its electrochemical gradient, the sugar or amino acid is, in a sense, "dragged" into the cell with it. The greater the electrochemical gradient for Na+, the more solute is pumped into the cell. - Mechanism of glucose transport fueled by a Na+ gradient: Binding of Na+ and glucose is cooperative: the binding of either solute induces a conformational change that increases the proteins affinity for the other solute. Since the Na+ concentration is much higher in the extracellular space than in the cytosol, glucose is more likely to bind to the transporter in the outward open state. The transition to the occluded occupied state occurs only when both Na+ and glucose are bound. The overall result is the net transport of both Na+ and glucose into the cell. *Because the occluded state is not formed when only one of the solutes is bound, the transporter switches conformation only when it is fully occupied or fully empty, thereby assuring strict coupling of the transport of Na+ and glucose.

properties of ion channels that distinguish them from aqueous pores

- Ion selectivity: permitting some inorganic ions to pass, but not others, suggesting that their pores must be narrow enough in places to force permeating ions into intimate contact with the walls of the channel so that only ions of appropriate size and charge can pass. The permeating ions have to shed most or all of their associated water molecules to pass, often in single file, through the narrowest part of the channel, which is called the selectivity filter; this limits their rate of passage. - Gated: ion channels are not continuously open, they open briefly and close again. With prolonged (chemical or electrical) stimulation, most ion channels go into a closed "desensitized," or "inactivated," state, in which they are refractory to further opening until the stimulus has been removed. In most cases, the gate opens in response to a specific stimulus.

STED (stimulated emission depletion) microscopy

- It creates super-resolution images by the selective deactivation of fluorophores, minimizing the area of illumination at the focal point, and thus enhancing the achievable resolution for a given system. - The resolution enhancement is essentially based on switching off the fluorescence of dye molecules by stimulated emission using intense laser light in the outer regions of the diffraction limited excitation focus. - This intense radiation causes almost all of the excited molecules to return to the ground state. Fluorescence from the remaining excited dye molecules in the center of the excitation focus is then detected and used to form the high resolution images.

diffusion of proteins in plasma membrane

- Like most membrane lipids, membrane proteins do not tumble (flip-flop) across the lipid bilayer, but they do rotate about an axis perpendicular to the plane of the bilayer (rotational diffusion). - In addition, many membrane proteins are able to move laterally within the membrane (lateral diffusion). - An experiment in which mouse cells were artificially fused with human cells to produce hybrid cells (heterokaryons) provided the first direct evidence that some plasma membrane proteins are mobile in the plane of the membrane. Two differently labeled antibodies (antibodies for mouse labeled with fluorescein and antibodies for human labeled with rhodamine) were used to distinguish selected mouse and human plasma membrane proteins. Although at first the mouse and human proteins were confined to their own halves of the newly formed heterokaryon, the two sets of proteins diffused and mixed over the entire cell surface in about half an hour

nucleocytoplasmic transport

- Many different classes of molecules and macromolecules are transported through nuclear pore complexes. - Small, uncharged molecules (<100 Da) can diffuse through the membrane of the nuclear envelope. - Bidirectional traffic occurs continuously between the cytosol and the nucleus - The proteins that function in the nucleus are selectively imported into the nuclear compartment form the cytosol, where they are made. - When gold particles of various sizes are injected into cells, those smaller than 9 nm in diameter can pass through NPCs by passive diffusion.

purpose of membrane

- Membranes allow the cytoplasm to maintain compartments with distinct environments - Organelles that are surrounded by membranes can maintain internal milieus that are different from the surrounding cytosol (e.g. pH, [Ca2+]

microsomes

- Microsomes represent small authentic versions of the ER, still capable of protein translocation, protein glycosylation, Ca2+ uptake and release, and lipid synthesis. - when tissues or cells are disrupted by homogenization, the ER breaks into fragments which reseal to form small closed vesicles called microsomes - easy to purify - When sedimenting a sample of microsomes, the two types of microsomes, smooth and rough, separate from each other on the basis of their different densities.

Translocation into mitochondria depends on signal sequences and protein translocators

- Mitochondrial proteins are first fully synthesized as mitochondrial precursor proteins in the cytosol and then translocated into mitochondria by a post-translational mechanism. - The signal sequences that direct precursor proteins into the mitochondrial matrix space are best understood. They all form an amphiphilic α helix, in which positively charged residues cluster on one side of the helix, while uncharged hydrophobic residues cluster on the opposite side. Specific receptor proteins that initiate protein translocation recognize this configuration rather than the precise amino acid sequence of the signal sequence. - Cytochrome oxidase is a large multiprotein complex located in the inner mitochondrial membrane, where it functions as the terminal enzyme in the electron-transport chain (discussed in Chapter 14). (A) The first 18 amino acids of the precursor to subunit IV of this enzyme serve as a signal sequence for import of the subunit into the mitochondrion. (B) When the signal sequence is folded as an α helix, the positively charged amino acids (red) are clustered on one face of the helix, while the nonpolar ones (green) are clustered primarily on the opposite face. Uncharged polar amino acids are shaded orange; nitrogen atoms on the side chains ofArg and Gln are colored blue. Signal sequences that direct proteins into the matrix space always have the potential to form such an amphiphilic α helix, which is recognized by specific receptor proteins on the mitochondrial surface. (C) The structure of a signal sequence (of alcohol dehydrogenase, another mitochondrial matrix enzyme), bound to an import receptor (gray), as determined by nuclear magnetic resonance. The amphiphilic α helix binds with its hydrophobic face toa hydrophobic groove in the receptor

fluorescent microscopy

- Most often used to detect specific proteins or other molecules in cells and tissues. - A very powerful and widely used technique is to couple fluorescent dyes to antibody molecules, which then serve as highly specific and versatile staining reagents that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix. - Two fluorescent dyes that have been commonly used for this purpose are fluorescein, which emits an intense green fluorescence when excited with blue light, and rhodamine, which emits deep red fluorescence when excited with green-yellow light

beta barrels

- Multipass membrane proteins that have their transmembrane segments arranged as β barrels rather than as α helices are comparatively rigid and therefore tend to form crystals readily when isolated. - β-barrel proteins are abundant in the outer membranes of bacteria, mitochondria, and chloroplasts.

nuclear import

- Nuclear import receptors recognize nuclear localization signals to initiate nuclear import of proteins containing the appropriate nuclear localization signal. - Each family member encodes a receptor protein that can bind and transport the subset of cargo proteins containing the appropriate nuclear localization signal. - Import concentrates specific proteins in the nucleus and increases order in the cell.

nuclear export

- Occurs through nuclear pore complexes - Relies on nuclear export signals on the macromolecules to be exported, as well as on complementary nuclear export receptors, or exportins. - These receptors bind to both the export signal and NPC proteins to guide their cargo through the NPC to the cytosol. - Many nuclear export receptors are structurally related to nuclear import receptors, and they are encoded by the same gene family of nuclear transport receptors, or karyopherins.

Ca2+ pump

- One of the best understood P-type ATPases - Found in the sarcoplasmic reticulum (SR) mem- brane of skeletal muscle cells - When an action potential depolarizes the muscle cell plasma membrane, Ca2+ is released into the cytosol from the SR through Ca2+-release channels, stimulating the muscle to contract. - The Ca2+ pump, which accounts for about 90% of the membrane protein of the SR, moves Ca2+ from the cytosol back into the SR. The endoplasmic reticulum of nonmuscle cells contains a similar Ca2+ pump, but in smaller quantities.

transfection approach

- One way to show that a signal sequence is sufficient to target a protein to a specific intracellular compartment is to create a fusion protein in which the signal sequence is attached by genetic engineering techniques to a protein that normally resides in the cytosol. After the cDNA encoding this protein is transfected into cells, the location of the fusion protein can be determined by immunostaining or cell fractionation. - If a GFP-fusion protein is designed to express, you can examine the transfected cells directly using a fluorescence microscope.

three common types of membrane lipids

- Phospholipids (major class) (i.e. phosphatidylserine) - Glycolipids (i.e. galactocerebroside) - Sterol (i.e. cholesterol)

hydropathy plots

- Plots measuring the hydrophobicity of proteins; used to determine potential membrane-spanning regions - The free energy needed to transfer successive segments of a polypeptide chain from a nonpolar solvent to water is calculated from the amino acid composition of each segment using data obtained from model compounds. This calculation is made for segments of a fixed size (usually around 20 amino acids), beginning with each successive amino acid in the chain. - The "hydropathy index" of the segment is plotted on the Y axis as a function of its location in the chain. - A positive value indicates that free energy is required for transfer to water (i.e., the segment is hydrophobic), and the value assigned is an index of the amount of energy needed. - Peaks in the hydropathy index appear at the positions of hydrophobic segments in the amino acid sequence. - In figure, glycophorin has a single membrane-spanning alpha helix and one corresponding peak while bacteriorhodopsin has seven membrane-spanning alpha helices and seven corresponding peaks.

equilibrium sedimentation

- Sediment depending on the buoyant density using a steep density gradient that contains a very high conc. Of sucrose or cesium chloride. - the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. - Each cell component begins to move down the gradient as in Figure 8-7A, but it eventually reaches a position where the density of the solution is equal to its own density. - At this point, the component floats and can move no farther. - A series of distinct bands is thereby produced in the centrifuge tube, with the bands closest to the bottom of the tube containing the components of highest buoyant density

velocity sedimentation

- Sediment depending on the size and shape, normally being described in terms of its sedimentation coefficient, or S value. - A finer degree of separation can be achieved by layering the homogenate in a thin band on top of a salt solution that fills a centrifuge tube. - When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate

polyribosome

- String of ribosomes simultaneously translating regions of the same mRNA strand during protein synthesis. - If the mRNA encodes a protein with an ER signal sequence, the polyribosome becomes attached to the ER membrane, directed there by the signal sequences on multiple growing polypeptide chains. The individual ribosomes associated with such an mRNA molecule can return to the cytosol when they finish translation and intermix with the pool of free ribosomes. The mRNA itself, however, remains attached to the ER membrane by a changing population of ribosomes, each transiently held at the membrane by the translocator

Auxiliary transport system in bacteria with double membranes

- The ABC transporters are located in the inner membrane, and an auxiliary mechanism operates to capture the nutrients and deliver them to the transporters - The solute diffuses through channel-forming proteins (porins) in the outer membrane and binds to a periplasmic substrate-binding protein, which undergoes a conformational change that enables it to bind to an ABC transporter in the plasma membrane. The ABC transporter then picks up the solute and actively transfers it across the plasma membrane in a reaction driven by ATP hydrolysis.

how does the signal-recognition particle direct the ER signal sequence?

- The SRP binds to both the exposed ER signal sequence and the ribosome, thereby inducing a pause in translation. The SRP receptor in the ER membrane, which is composed of two different polypeptide chains, binds the SRP-ribosome complex and directs it to the translocator. - Because one of the SRP proteins and both chains of the SRP receptor contain GTP-binding domains, it is thought that conformational changes that occur during cycles of GTP binding and hydrolysis ensure that SRP release occurs only after the ribosome has become properly engaged with the translocator in the ER membrane.

Conventional vs confocal fluorescence microscopy

- The confocal microscope has been used to resolve the structure of numerous complex three-dimensional objects including the networks of cytoskeletal fibers in the cytoplasm and the arrangements of chromosomes and genes in the nucleus. - The conventional, unprocessed image is blurred by the presence of fluorescent structures above and below the plane of focus. - In the confocal image, this out-of-focus information is removed, resulting in a crisp optical section of the cells in the embryo.

cell membranes as selective barriers

- The contents of a cell can be separated from the environment by cell membranes. - Its permeability properties ensure that essential molecules such as glucose, amino acids, and lipids readily enter the cell, metabolic intermediates remain in the cell, and waste compounds leave the cell. - The plasma membrane encloses the cell, defines its boundaries, and maintains the essential differences between the cytosol and the extracellular environment while the internal membrane encloses an intracellular compartment.

chemical composition of membranes

- The core of the membrane consists of a sheet of lipids arranged in a bilayer. The lipid bilayer serves primarily as a structural backbone of the membrane and provides the barrier that prevents random movements of water-soluble materials through the membrane. - The proteins of the membrane carry out most of the specific functions and give different membranes their individual characteristics. - The ratio of lipid to protein in a membrane varies, depending on the type of cellular membrane (plasma vs. endoplasmic reticulum vs. Golgi), the type of organism (bacterium vs. plant vs. animal), and the type of cell (cartilage vs. muscle vs. liver).

glycolysation of proteins synthesized in the rough ER

- The covalent addition of oligosaccharides to proteins is one of the major biosynthetic functions of the ER. About half of the soluble and membrane-bound proteins that are processed in the ER—including those destined for transport to the Golgi apparatus, lysosomes, plasma membrane, or extracellular space—are glycoproteins that are modified in this way. - During the most common form of protein glycosylation in the ER, a pre- formed precursor oligosaccharide (composed of N-acetylglucosamine, mannose, and glucose, and containing a total of 14 sugars) is transferred en bloc to proteins. Because this oligosaccharide is transferred to the side-chain NH2 group of an asparagine in the protein, it is said to be N-linked or asparagine-linked. The transfer is catalyzed by a membrane-bound enzyme complex, an oligosaccharyl transferase, which has its active site exposed on the lumenal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. A special lipid molecule called dolichol anchors the precursor oli- gosaccharide in the ER membrane. The precursor oligosaccharide is transferred to the target asparagine in a single enzymatic step immediately after that amino acid has reached the ER lumen during protein translocation. The precursor oligo- saccharide is linked to the dolichol lipid by a high-energy pyrophosphate bond, which provides the activation energy that drives the glycosylation reaction.

Cis-double bonds in hydrocarbon chains affect their packing

- The double bonds make it more difficult to pack the chains together, thereby making the lipid bilayer more difficult to freeze. - Because the hydrocarbon chains of unsaturated lipids are more spread apart, lipid bilayers containing them are thinner than bilayers formed exclusively from saturated lipids. - The fluidity of a lipid bilayer depends on its composition. - A shorter chain length reduces the tendency of the hydrocarbon tails to interact with one another, in both the same and opposite monolayer, and cis-double bonds produce kinks in the chains that make them more difficult to pack together, so that the membrane remains fluid at lower temperatures

four major color classes of GFP derivatives

- The fluorescence properties are dependent on the arrangement and three-dimensional structure of amino acid residues surrounding the chromophore. - By changing one or more of three residues of GFP, you can change the fluorescence of GFP - derivatives are BFP, CFP, EGFP, EYFP

lipid bilayer

- The lipid bilayers in many cell membranes contain phospholipids, cholesterol and glycolipids. Eukaryotic plasma membranes contain especially large amounts of cholesterol - up to one molecule for every phospholipid molecule. - Can be considered a two-dimensional fluid. - Lipid molecules rapidly exchange places with their neighbors within a monolayer (~107 times per second). - This gives rise to a rapid lateral diffusion, with a diffusion coefficient (D) of about 10-8 cm2/sec, which means that an average lipid molecule diffuses the length of a large bacterial cell (~2 μm) in about 1 second. - These studies have also shown that individual lipid molecules rotate very rapidly about their long axis and have flexible hydrocarbon chains.

asymmetry of the lipid bilayer

- The lipid compositions of the two monolayers of the lipid bilayer in many membranes are strikingly different. - Lipid asymmetry is functionally important, especially in converting extra- cellular signals into intracellular ones. - Many cytosolic proteins bind to specific lipid head groups found in the cytosolic monolayer of the lipid bilayer.

phosphoglycerides

- The main phospholipids in most animal cell membranes. - Have a three-carbon glycerol backbone. - Two long-chain fatty acids are linked through ester bonds to adjacent carbon atoms of the glycerol, and the third carbon atom of the glycerol is attached to a phosphate group, which in turn is linked to one of several types of head group.

makeup of the cell

- The membrane-enclosed compartments together occupy nearly half the volume of a cell and a large amount of intracellular membrane is required to make them. - The membrane-enclosed organelles are packed tightly in the cytoplasm, and, in terms of area and mass, the plasma membrane is only a minor membrane in most eukaryotic cells

gangliosides

- The most complex of the glycolipids. - Contain oligosaccharides with one or more sialic acid moieties, which give gangliosides a net negative charge. - The most abundant of the more than 40 different gangliosides that have been identified are in the plasma membrane of nerve cells, where gangliosides constitute 5-10% of the total lipid mass; they are also found in much smaller quantities in other cell types.

Bacteria and mitochondria use similar mechanisms to insert porins into their outer membrane

- The outer mitochondrial membrane, like the outer membrane of Gram-negative bacteria, contains abundant pore-forming proteins called porins and is thus freely permeable to inorganic ions and metabolites (but not to most proteins). - After translocation through the TOM complex, b-barrel proteins (porins) bind to chaperones in the intermembrane space. The SAM complex then inserts the unfolded polypeptide chain into the outer membrane.

peripheral proteins

- The proteins of a membrane that are not embedded in the lipid bilayer; they are appendages loosely bound to the surface of the membrane. - attached to the membrane only by noncovalent interactions with other membrane proteins

how do hydrophilic and hydrophobic molecules interact differently with water?

- The shape and amphiphilic nature of the phospholipid molecules cause them to form bilayers spontaneously in aqueous environments. -Hydrophilic molecules dissolve readily in water because they contain charged groups or uncharged polar groups that can form either favorable electrostatic interactions or hydrogen bonds with water molecules - Hydro- phobic molecules, by contrast, are insoluble in water because all, or almost all, of their atoms are uncharged and nonpolar and therefore cannot form energetically favorable interactions with water molecules. If dispersed in water, they force the adjacent water molecules to reorganize into icelike cages that surround the hydrophobic molecule - Acetone readily dissolves in water, because acetone is polar, it can form favorable electrostatic interactions with water molecules, which are also polar. - 2-methylpropane is virtually insoluble in water, because it is entirely hydrophobic, it cannot form favorable interactions with water, it would force adjacent water molecules to reorganize into cage-like structures, which is more highly ordered than the surrounding water and requires energy for its formation

lipid rafts

- There has been a long debate among cell biologists about whether the lipid molecules in the plasma membrane of living cells similarly segregate into these specialized domains - Although many lipids and membrane proteins are not distributed uniformly, large-scale lipid phase segregations are rarely seen in living cell membranes. - Instead, specific membrane proteins and lipids are seen to concentrate in a more temporary, dynamic fashion facilitated by protein- protein interactions that allow the transient formation of specialized membrane regions.

how does Ran drive nuclear transport?

- Through the gradient of two conformational forms of Ran - Docking of nuclear import receptors to FG-repeats on the cytosolic side of the NPC, for example, occurs whether or not these receptors are loaded with appropriate cargo. - Import receptors, facilitated by FG-repeat binding, then enter the channel. - If they reach the nuclear side of the pore com- plex, Ran-GTP binds to them, and, if the receptors arrive loaded with cargo mol- ecules, the Ran-GTP binding causes the receptors to release their cargo. - Because the Ran-GDP in the cytosol does not bind to import (or export) receptors, unloading occurs only on the nuclear side of the NPC. In this way, the nuclear localization of Ran-GTP creates the directionality of the import process. - Having discharged its cargo in the nucleus, the empty import receptor with Ran-GTP bound is transported back through the pore complex to the cytosol. There, Ran-GAP triggers Ran-GTP to hydrolyze its bound GTP, thereby converting it to Ran-GDP, which dissociates from the receptor. The receptor is then ready for another cycle of nuclear import. - Nuclear export occurs by a similar mechanism, except that Ran-GTP in the nucleus promotes cargo binding to the export receptor, rather than promoting cargo dissociation. Once the export receptor moves through the pore to the cyto- sol, it encounters Ran-GAP, which induces the receptor to hydrolyze its GTP to GDP. As a result, the export receptor releases both its cargo and Ran-GDP in the cytosol. Free export receptors are then returned to the nucleus to complete the cycle

active transport

- Transport in which certain solutes are actively pumped across their membrane "uphill," against their electrochemical gradients. - Mediated by transporters whose pumping activity is directional because it is tightly coupled to a source of metabolic energy, such as an ion gradient or ATP hydrolysis.

transcellular transport

- Transport of materials through the cell; requires interaction with the cytoplasm and may require transport proteins - Occurs due to nonuniform distribution of transporters in the cell's plasma membrane - Na+-linked symporters located in the apical domain of the plasma membrane actively transport nutrients into the cell, building up substantial concentration gradients for these solutes across the plasma membrane. Na+-independent transport proteins in the basal and lateral (basolateral) domain allow the nutrients to leave the cell passively down these concentration gradients.

V-type pump

- Turbine-like protein machines, constructed from multiple different subunits. - The V-type proton pump transfers H+ into organelles such as lysosomes, synaptic vesicles, and plant or yeast vacuoles (V=vacuolar), to acidify the interior of these organelles.

tandem mass spectrometry (MS/MS)

- Using two mass analyzers in tandem to determine the amino acid sequence of a single protein contained within a mixture of proteins - Tandem mass spectrometry typically involves two mass analyzers separated by a collision chamber containing an inert, high-energy gas. - The electric field of the first mass analyzer is adjusted to select a specific peptide ion, called a precursor ion, which is then directed to the collision chamber. - Collision of the peptide with gas molecules causes random peptide fragmentation, primarily at peptide bonds, resulting in a highly complex mixture of fragments containing one or more amino acids from throughout the original peptide. - The second mass analyzer is then used to measure the masses of the fragments (called product or daughter ions). - With computer assistance, the pattern of fragments can be used to deduce the amino acid sequence of the original peptide.

permeability of lipid bilayers

- Virtually any molecule will diffuse across a protein-free lipid bilayer down its concentration gradient. - The rate of diffusion, however, varies enormously, depending partly on the size of the molecule but mostly on its relative hydrophobicity (solubility in oil). - In general, the smaller the molecule and the more hydrophobic, or nonpolar, it is, the more easily it will diffuse across a lipid bilayer. - Small nonpolar molecules, such as O2 and CO2, readily dissolve in lipid bilayers and therefore diffuse rapidly across them. - Small uncharged polar mole- cules, such as water or urea, also diffuse across a bilayer, albeit much more slowly. - By contrast, lipid bilayers are essentially imper- meable to charged molecules (ions), no matter how small: the charge and high degree of hydration of such molecules prevents them from entering the hydrocarbon phase of the bilayer. - The rate of flow of a solute across the lipid bilayer is directly proportional to the difference in its concentration on the two sides of the membrane.

aquaporins

- Water channels embedded in the plasma membrane of prokaryotic and eukaryotic cells to allow water to move more rapidly. - Particularly abundant in animal cells that must transport water at high rates, such as the epithelial cells of the kidney or exocrine cells that must transport or secrete large volumes of fluids, respectively. - These cells are organized into epithelial sheets in which their apical plasma membrane faces the lumen of the duct. - Ion pumps and channels situated in the basolateral and apical plasma membrane move ions (mostly Na+ and Cl-) into the ductal lumen, creating an osmotic gradient between the surrounding tissue and the duct. - Water molecules rapidly follow the osmotic gradient through aquaporins that are present in high concentrations in both the apical and basolateral membranes.

signal hypothesis

- When signal sequences were first discovered, the mRNA encoding a secreted protein was translated by ribosomes in vitro. When microsomes were omitted from this cell-free system, the protein synthesized was slightly larger than the normal secreted protein. In the presence of microsomes derived from the rough ER, however, a protein of the correct size was produced. - According to the signal hypothesis, the size difference reflects the initial presence of a signal sequence that directs the secreted protein to the ER membrane and is then cleaved off by a signal peptidase in the ER membrane before the polypeptide chain has been completed.

insertion mechanism of ER tail-anchored proteins

- When such a tail-anchored protein inserts into the ER membrane from the cytosol, only a few amino acids that follow the transmembrane α helix on its C-terminal side are translocated into the ER lumen, while most of the protein remains in the cytosol. Because of the unique position of the transmembrane α helix in the protein sequence, translation terminates while the C-terminal amino acids that will form the transmembrane α helix have not yet emerged from the ribosome exit tunnel. Recognition by SRP is therefore not possible. - In this post-translational pathway, a soluble pre-targeting complex captures the hydrophobic C-terminal α helix after it emerges from the ribosomal exit tunnel and loads it onto the Get3 ATPase. The resulting complex is targeted to the ER membrane by interaction with the Get1- Get2 receptor complex that functions as a membrane protein insertion machine. After Get3 hydrolyzes its bound ATP, the tail-anchored protein is released from the receptor and inserted into the ER membrane. ADP release and renewed ATP binding recycles Get3 back to the cytosol.

SDS polyacrylamide gel electrophoresis

- a complex mixture of proteins is fractionated into a series of discrete protein bands arranged in order of molecular weight - It uses a highly cross-linked gel of polyacrylamide as the inert matrix through which the proteins migrate. The gel is prepared by polymerization of monomers; the pore size of the gel can be adjusted so that it is small enough to retard the migration of the protein molecules of interest. The proteins are dissolved in a solution that includes a powerful negatively charged detergent, sodium dodecyl sulfate, or SDS. Because this detergent binds to hydrophobic regions of the protein molecules, causing them to unfold into extended polypeptide chains, the individual protein molecules are released from their associations with other proteins or lipid molecules and rendered freely soluble in the detergent solution. In addition, a reducing agent such as β-mercaptoethanol (see Figure 8-12) is usually added to break any S-S linkages in the proteins, so that all of the constituent polypeptides in multisubunit proteins can be analyzed separately. - Proteins of the same size tend to move through the gel with similar speeds because (1) their native structure is completely unfolded by the SDS, so that their shapes are the same, and (2) they bind the same amount of SDS and therefore have the same amount of negative charge. Larger proteins, with more charge, are subjected to larger electrical forces but also to a larger drag.

two-dimensional gel electrophoresis

- a laboratory method that separates proteins according to their isoelectric points and molecular weights - In the first step, the proteins are separated by their intrinsic charges. - The polypeptide chains are then separated in a pH gradient by a procedure called isoelectric focusing, which takes advantage of the variation in the net charge on a protein molecule with the pH of its surrounding solution. - In the second step, the narrow tube gel containing the separated proteins is again subjected to electrophoresis but in a direction that is at a right angle to the direction used in the first step. This time SDS is added, and the proteins separate according to their size.

Green fluorescent protein (GFP)

- a protein that exhibits bright green fluorescence when exposed to blue light - GFP consists of eleven β strands that form the staves of a barrel. Buried within the barrel is the active chromophore (dark green) that is formed post-translationally from the protruding side chains of three amino acid residues.

membrane proteins

- amphiphilic, having hydrophobic and hydrophilic regions - perform most of the membrane's specific tasks and therefore give each type of cell membrane its characteristic functional properties. - accordingly, the amounts and types of proteins in a membrane are highly variable. - Exposed at only one side of the membrane; anchored to the cytosolic surface by an amphiphilic a helix that partitions into the cytosolic monolayer of the lipid bilayer through the hydrophobic face of the helix, attached to the bilayer solely by a covalently attached lipid chain-either a fatty acid chain or a prenyl group-in the cytosolic monolayer or, via an oligosaccharide linker, to phosphatidylinositol in the noncytosolic monolayer-called a glycosylphosphatidylinositol (GPI) anchor.

lipid droplet

- an excess of lipids found in most cells from where they can be retrieved as building block for membrane synthesis or as a food source - most cells have many smaller lipid droplets, the number and size varying with the cell's metabolic state. - fatty acids can be liberated from lipid droplets on demand and exported to other cells through the bloodstream. - they are unique organelles in that they are surrounded by a single monolayer of phospholipids, which contains a large variety of proteins.

mitochondria and chloroplasts

- both double-membrane enclosed organelles - mitochondria: synthesize ATP using energy derived from electron transport and oxidative phosphorylation - chloroplast: synthesize ATP using energy derived from photosynthesis - Most of their proteins are encoded in the cell nucleus and imported from the cytosol. - New mitochondria and chloroplasts are produced by the growth of preexisting organelles, followed by fission.

fluorescence resonance energy transfer (FRET)

- can be used to determine whether (and when) two proteins interact inside a cell. - the proteins are first produced as fusion proteins attached to different color variants of green fluorescent protein (GFP).

how do mammalian cells import most of their proteins into the ER?

- co-translationally; they begin to import most proteins into the ER before complete synthesis of the polypeptide chain - the ribosome that is synthesizing the protein is attached directly to the ER membrane, enabling one end of the protein to be translocated into the ER while the rest of the polypeptide chain is being synthesized. These membrane-bound ribosomes coat the surface of the ER, creating regions termed rough endoplasmic reticulum.

E. coli OmpA protein

- contains a beta barrel - The E. coli OmpA protein serves as a receptor for a bacterial virus. - The E.coli OMPLA protein is an enzyme (a lipase) that hydrolyzes lipid molecules. The amino acids that catalyze the enzymatic reaction (shown in red) protrude from the outside surface of the barrel. - A porin from the bacterium Rhodobacter capsulatus forms a waterfilled pore across the outer membrane. The diameter of the channel is restricted by loops (shown in blue) that protrude into the channel. - The E. coli FepA protein transports iron ions. - The inside of the barrel is completely filled by a globular protein domain (shown in blue) that contains an iron-binding site (not shown). - This domain is thought to change its conformation to transport the bound iron, but the molecular details of the changes are not known.

cortical cytoskeletal filaments

- cortical cytoskeletal filaments provide diffusion barriers that divide the membrane into small domains, or corrals - high speed, single-particle tracking demonstrated that individual protein molecules diffuse within a tightly delimited membrane domain and only infrequently escape into a neighboring domain

signal sequences

- direct proteins to the correct cell addresses - Most protein sorting signals reside in a stretch of amino acid sequence, typically 15-60 residues long. These signal sequences are often found at the N-terminus; specialized signal peptidases remove the signal sequence from the finished protein once the sorting process is complete. Signal sequences can also be internal stretches of amino acids, which remain part of the protein. In some cases, sorting signals are composed of multiple internal amino acid sequences that form a specific three-dimensional arrangement of atoms on the protein's surface, called a signal patch.

nuclear envelope

- encloses the DNA and defines the nuclear compartment - consists of two concentric membranes, which are penetrated by nuclear pore complexes

how did modern eukaryotic cells evolve?

- from a symbiosis - Intake of aerobic bacterium is similar to phagocytosis -Membrane pinches off after intake of bacterium, membrane from bacterium is removed, and the plasma membrane of the host becomes plasma membrane of bacterium - Explains why chloroplasts and mitochondria share homology with bacteria

cortical cytoskeleton

- gives membranes mechanical strength and restricts membrane protein diffusion - The cortical cytoskeletal network restricts diffusion of not only the plasma membrane proteins that are directly anchored to it. Because the cytoskeletal filaments are often closely apposed to the cytosolic surface of the plasma membrane, they can form mechanical barriers that obstruct the free diffusion of proteins in the membrane.

polypeptide chains in transmembrane proteins

- in most transmembrane proteins, the polypeptide chain crosses the lipid bilayer in an a-helical conformation - 3.6 residues per turn - The domains of the transmembrane protein are separated by the membrane-spanning segments of the polypeptide chain, which contact the hydrophobic environment of the lipid bilayer and are composed largely of amino acids with nonpolar side chains. Because the peptide bonds themselves are polar and because water is absent, all peptide bonds in the bilayer are driven to form hydrogen bonds with one another. The hydrogen-bonding between peptide bonds is maximized if the polypeptide chain forms a regular α helix as it crosses the bilayer, and this is how most membrane-spanning segments of polypeptide chains traverse the bilayer

photoactivation

- light-induced activation of an inert molecule to an active state - allows detailed observations of proteins within cells involves synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot of light on it.

biological role of lipids

- major components of membranes. - serve as fuel molecules. - signal molecules and messages in signal transduction pathways (Ch.15, eg. Diacyglycerol, IP3 and etc).

transmembrane proteins

- membrane proteins that extend through the lipid bilayer - Extend across the bilayer as (1) a single a helix, (2) & (3) multiple a helices. - Some of these "single-pass" and "multipass" proteins have a covalently attached fatty acid chain inserted in the cytosolic lipid monolayer (1). - Some membrane proteins cross the lipid bilayer via a b barrel (a rolled-up b sheet, not shown here). - their hydrophobic regions pass through the membrane and interact with the hydrophobic tails of the lipid molecules in the interior of the bilayer, where they are sequestered away from water. - their hydrophilic regions are exposed to water on either side of the membrane. - The covalent attachment of a fatty acid chain that inserts into the cytosolic monolayer of the lipid bilayer increases the hydrophobicity of some of these transmembrane proteins.

glycosylated membrane proteins

- most transmembrane proteins in animal cells are glycosylated (addition of carbohydrate to a hydroxyl or other functional group) - the sugar residues of glycoproteins are added in the lumen of the ER and the Golgi apparatus. - The oligosaccharide chains are always present on the noncytosolic side of the membrane. - The polypeptide chain traverses the lipid bilayer as a right-handed a helix and that the oligosaccharide chains and disulfide bonds are all on the noncytosolic surface of the membrane. - The sulfhydryl groups in the cytosolic domain of the protein do not normally form disulfide bonds because the reducing environment in the cytosol maintains these groups in their reduced (-SH) form.

fluorescence recovery after photobleaching (FRAP)

- one way to exploit GFP fused to a protein of interest - one uses a strong focused beam of light from a laser to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time. - The fluorescence intensity recovers as the bleached molecules diffuse away and unbleached molecules diffuse into the irradiated area. - can deliver valuable quantitative data about a protein's kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins - The diffusion coefficient is calculated from a graph of the rate of recovery: the greater the diffusion coefficient of the membrane protein, the faster the recovery. - done with a confocal microscope

nuclear pore complex

- perforate the nuclear envelope in all eukaryotes - each NPC is composed of a set of approximately 30 different proteins (nucleoporins) - Each nucleoporin is present in multiple copies, resulting in 500-1000 protein mol- ecules in the fully assembled NPC

The major features of eukaryotic cells

- plasma membrane - nucleus - cytoplasm - ribosomes - cell wall (in some groups) - complex structures for locomotion like cilia and flagella - membrane-bound organisms like mitochondria, chloroplasts, golgi apparatus, etc. - centrioles in some

how do mammalian cells import proteins into mitochondria, chloroplasts, nuclei, and peroxisomes?

- post-translationally; cytosolic ribosomes complete the synthesis of a protein and release it prior to post-translational translocation.

free ribosomes

- synthesize all other proteins encoded by the nuclear genome - unattached to any membrane - membrane-bound and free ribosomes are structurally and functionally identical.

lenses

- the objective lens collects a cone of light rays to create an image. - the condenser lens focuses a cone of light rays onto each point of the specimen

neuromuscular transmission

- the process in which a nerve impulse stimulates a muscle cell to contract requires the sequential activation of at least five different sets of ion channels 1. The process is initiated when a nerve impulse reaches the nerve terminal and depolarizes the plasma membrane of the terminal. The depolarization transiently opens voltage-gated Ca2+ channels in this presynaptic mem- brane. As the Ca2+ concentration outside cells is more than 1000 times greater than the free Ca2+ concentration inside, Ca2+ flows into the nerve terminal. The increase in Ca2+ concentration in the cytosol of the nerve terminal triggers the local release of acetylcholine by exocytosis into the synaptic cleft. 2. The released acetylcholine binds to acetylcholine receptors in the muscle cell plasma membrane, transiently opening the cation channels associated with them. The resulting influx of Na+ causes a local membrane depolarization. 3. The local depolarization opens voltage-gated Na+ channels in this mem- brane, allowing more Na+ to enter, which further depolarizes the mem- brane. This, in turn, opens neighboring voltage-gated Na+ channels and results in a self-propagating depolarization (an action potential) that spreads to involve the entire plasma membrane. 4. The generalized depolarization of the muscle cell plasma membrane activates voltage-gated Ca2+ channels in the transverse tubules (T tubules of this membrane. 5. This in turn causes Ca2+-release channels in an adjacent region of the sarcoplasmic reticulum (SR) membrane to open transiently and release Ca2+ stored in the SR into the cytosol. The T-tubule and SR membranes are closely apposed with the two types of channel joined together in a special- ized structure, in which activation of the voltage-sensitive Ca2+ channel in the T-tubule plasma membrane causes a channel conformational change that is mechanically transmitted to the Ca2+-release channel in the SR membrane, opening it and allowing Ca2+ to flow from the SR lumen into the cytoplasm (see Figure 16-35). The sudden increase in the cytosolic Ca2+ concentration causes the myofibrils in the muscle cell to contract.

two main classes of membrane transport proteins

- transporters and channels - A transporter alternates between two conformations, so that the solute-binding site is sequentially accessible on one side of the bilayer and then on the other. - A channel protein forms a water-filled pore across the bilayer through which specific solutes can diffuse.

indirect immunocytochemistry

- using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it in order to detect and assay specific molecules in cells - very sensitive because many molecules of the secondary antibody recognize each primary antibody. - the secondary antibody is covalently coupled to a marker molecule that makes it readily detectable.

how do some membrane proteins attach to the membrane?

- via a fatty acid chain or a prenyl group - The covalent attachment of either type of lipid can help localize a water-soluble protein to a membrane after its synthesis in the cytosol. - A myristic acid is attached via an amide linkage to an N-terminal glycine. - A palmitic acid is attached via a thioester linkage to a cysteine. - A prenyl group (either farnesyl or a longer geranylgeranyl group) is attached via a thioether linkage to a cysteine residue that is initially located four residues from the protein's C-terminus. After prenylation, the terminal three amino acids are cleaved off, and the new C-terminus is methylated before insertion of the anchor into the membrane (not shown).

Membrane proteins can be solubilized and purified in detergents

-Only agents that disrupt hydrophobic associations and destroy the lipid bilayer can solubilize membrane proteins. - The most useful of these for the membrane biochemist are detergents, which are small amphiphilic molecules of variable structure. - Detergent molecules are amphiphilic; and because they are cone-shaped, they form micelles rather than bilayers. Detergent micelles have irregular shapes. Due to packing constraints, the hydrophobic tails are partially exposed to water. - At low concentration, detergent molecules are monomeric in solution. As their concentration is increased beyond the critical micelle concentration (CMC), some of the detergent molecules form micelles. - A mild nonionic detergent solubilizes membrane proteins. - The detergent disrupts the lipid bilayer and brings the proteins into solution as protein-lipid-detergent complexes. - The phospholipids in the membrane are also solubilized by the detergent.

three methods of active transport

1. Coupled transporters harness the energy stored in concentration gradients to couple the uphill transport of one solute across the membrane to the downhill transport of another. 2. ATP-driven pumps couple uphill transport to the hydrolysis of ATP. 3. Light-or redox-driven pumps, which are known in bacteria, archaea, mitochondria, and chloroplasts, couple uphill transport to an input of energy from light, as with bacteriorhodopsin, or from a redox reaction, as with cytochrome c oxidase.

image deconvolution method

A computational approach that sharps the digital images. The computer program is used to remove as much the blurring part of the images as possible, turning the blurred 3D image into a series of clean optical sections

resolving power

A measure of the clarity of an image; the ability of an optical instrument to show two objects as separate.

how is the ER signal sequence guided to the ER membrane?

A signal-recognition particle and an SRP receptor

fluorescence loss in photobleaching (FLIP)

A small area of membrane is irradiated continuously, and fluorescence is measured in a separate area. Fluorescence in the second area progressively decreases as fluorescent proteins diffuse out and bleached molecules diffuse in; eventually, all of the fluorescent protein molecules are bleached, as long as they are mobile and not permanently anchored to the cytoskeleton or extracellular matrix.

affinity chromatography

Affinity chromatography (Figure 8-9C) takes advantage of the biologically important binding interactions that occur on protein surfaces. If a substrate molecule is covalently coupled to an inert matrix such as a polysaccharide bead, the enzyme that operates on that substrate will often be specifically retained by the matrix and can then be eluted (washed out) in nearly pure form. Likewise, short DNA oligonucleotides of a specifically designed sequence can be immobilized in this way and used to purify DNA-binding proteins that normally recognize this sequence of nucleotides in chromosomes. Alternatively, specific antibodies can be coupled to a matrix to purify protein molecules recognized by the antibodies.

electrochemical gradient

An electrochemical gradient can work additively to increase the driving force on an ion across the membrane (middle) or can work against each other (right).

integration of a single-pass transmembrane protein with an internal signal sequence into the ER membrane

An internal ER signal sequence (also binds to the SRP) that functions as start transfer signal can bind to the translocator in one of two different ways, leading to a membrane protein that has either its C-terminus (pathway A) or its N-terminus (pathway B) in the ER lumen. Proteins are directed into either pathway by features in the polypeptide chain flanking the internal start transfer sequence.

formation of domains of different compositions in lipid bilayers

Because a lipid bilayer is a two-dimensional fluid, we might expect most types of lipid molecules in it to be well mixed and randomly distributed in their own monolayer. The van der Waals attractive forces between neighboring hydrocarbon tails are not selective enough to hold groups of phospholipid molecules together. With certain lipid mixtures in artificial bilayers, however, one can observe phase segregations in which specific lipids come together in separate domains.

ER tail-anchored proteins

C-terminal transmembrane, hydrophobic alpha helix that anchors many important membrane proteins in the membrane.

what factors affect the melting temperature of the lipid bilayer?

Chain length and degree of unsaturation of fatty acids

electron density in transmission electron microscopy

Electron density depends on the atomic number of the atoms that are present: the higher the atomic number the more electrons are scattered and the darker that part of the image.

commonly used marker molecules

Fluorescent dyes: for fluorescence microscopy. Enzyme horseradish peroxidase: for either conventional light microscopy or electron microscopy. Colloidal gold spheres: for electron microscopy. Enzyme alkaline phosphatase or peroxidase: for biochemical detection.

cells can confine proteins and lipids to specific domains within a membrane

Four ways of restricting the lateral mobility of specific plasma membrane proteins via protein-protein interactions: (A) The proteins can self-assemble into large aggregates (e.g. bacteriorhodopsin in the purple membrane of Halobacterium); (B)& (C) The proteins can be tethered by interactions with assemblies of macromolecules (B) outside or (C) inside the cell; (D) The proteins can interact with proteins on the surface of another cell.

An example of using mild nonionic detergents for solubilizing, purifying, and reconstituting functional membrane protein systems.

Functional Na+-K+ pump molecules are purified and incorporated into phospholipid vesicles.

What molecule performs most functions in the cell?

Functions within cells are mainly carried out by proteins, alone or complexed with other proteins, RNA and lipids.

gel-filtration chromatography

Gel-filtration columns, which separate proteins according to their size, are packed with tiny porous beads: molecules that are small enough to enter the pores linger inside successive beads as they pass down the column, while larger molecules remain in the solution flowing between the beads and therefore move more rapidly, emerging from the column first.

asymmetry of lipid bilayer in human red blood cells

In the human red blood cell (erythrocyte) membrane, for example, almost all of the phospholipid molecules that have choline—(CH3)3N+CH2CH2OH—in their head group (phosphatidylcholine and sphingomyelin) are in the outer monolayer, whereas almost all that contain a terminal primary amino group (phosphatidylethanolamine and phosphatidylserine) are in the inner monolayer

integration of a double-pass membrane protein with an internal signal sequence into the ER membrane

In this protein, an internal ER signal sequence acts as a start-transfer signal and initiates the transfer of the C-terminal part of the protein. At some point after a stoptransfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane.

how is a single-pass transmembrane protein with a cleaved ER signal sequence integrated into the ER membrane?

In this protein, the co-translational translocation process is initiated by an N-terminal ER signal sequence (red) that functions as a start-transfer signal. In addition to this start-transfer sequence, however, the protein also contains a stop-transfer sequence (orange).When the stop-transfer sequence enters the translocator and interacts with a binding site, the translocator changes its conformation and discharges the protein laterally into the lipid bilayer. * In this figure, the ribosomes have been omitted for clarity.

Pumping cycle of Ca2+ pump

Ion pumping proceeds by a series of stepwise conformational changes in which movements of the pump's three cytosolic domains are mechanically coupled to movements of the transmembrane α helices. Helix movement opens and closes passageways through which Ca2+ enters from the cytosol and binds to the two centrally located Ca2+ binding sites. The two Ca2+ then exit into the SR lumen and are replaced by two H+, which are transported in the opposite direction. The Ca2+ -dependent phosphorylation and H+-dependent dephosphorylation of aspartic acid are universally conserved steps in the reaction cycle of all P-type pumps: they cause the conformational transitions to occur in an orderly manner, enabling the proteins to do useful work.

ion-exchange chromatography

Ion-exchange columns are packed with small beads that carry either a positive or a negative charge, so that proteins are fractionated according to the arrangement of charges on their surface.

light microscope

Light is focused on the specimen by lenses in the condenser. A combination of objective lenses, tube lenses, and eyepiece lenses is arranged to focus an image of the illuminated specimen in the eye.

composition of membrane lipids

Membrane lipids are amphiphilic (also called amphipathic) molecules containing a hydrophilic (water-loving) and a hydrophobic (water-fearing) moiety.

liposomes

Membrane-bound droplets that form when lipids are added to water.

examples of antiporters

NCX (Na+/Ca2+) exchanger, NHE (Na+/H+ exchanger), AE (Cl-/HCO3- exchanger)

synapse

Neuronal signals are transmitted from cell to cell at these specialized sites of contact

formation of lipid droplets

Neutral lipids are deposited between the two monolayers of the endoplasmic reticulum membrane. There, they aggregate into a 3D droplet, which buds and pinches off from the ER membrane as a unique organelle, surrounded by a single monolayer of phospholipids and associated proteins.

fluorescence-activated sorting

One of the most sophisticated cell-separation techniques uses an antibody coupled to a fluorescent dye to label specific cells. An antibody is chosen that specifically binds to the surface of only one cell type in the tissue. The labeled cells can then be separated from the unlabeled ones in a fluorescence-activated cell sorter. In this remarkable machine, individual cells traveling single file in a fine stream pass through a laser beam, and the fluorescence of each cell is rapidly measured. A vibrating nozzle generates tiny droplets, most containing either one cell or no cells. The droplets containing a single cell are automatically given a positive or a negative charge at the moment of formation, depending on whether the cell they contain is fluorescent; they are then deflected by a strong electric field into an appropriate container. Occasional clumps of cells, detected by their increased light scattering, are left uncharged and are discarded into a waste container.

four major phospholipids in mammalian plasma membranes

Phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and sphingolipids make up more than half the mass of lipid in most mammalian cell membranes

Three major divisions (domains) of the living world

Prokaryotes (bacteria, archaea), and eukaryotes

protein traffic

Proteins can move from one compartment to another by gated transport (red), protein translocation (blue), or vesicular transport (green). The sorting signals that direct a given protein's movement through the system, and determine its eventual location in the cell, are contained in each protein's amino acid sequence. The journey begins with the synthesis of a protein on a ribosome in the cytosol and, for many proteins, terminates when the protein reaches its final destination. Other proteins shuttle back and forth between the nucleus and cytosol. At each intermediate station (boxes), a decision is made as to whether the protein is to be retained in that compartment or transported further. A sorting signal may direct either retention in or exit from a compartment.

cell fractionation by centrifugation

Repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components. In general, the smaller the subcellular component, the greater the centrifugal force required to sediment it.

what factor affects the location of a protein?

Some proteins contain both nuclear localization signals and nuclear export sig- nals. These proteins continually shuttle back and forth between the nucleus and the cytosol. The relative rates of their import and export determine the steady- state localization of such shuttling proteins: if the rate of import exceeds the rate of export, a protein will be located mainly in the nucleus; conversely, if the rate of export exceeds the rate of import, a protein will be located mainly in the cytosol. Thus, changing the rate of import, export, or both, can change the location of a protein.

fluid mosaic model

Structural model of the plasma membrane where molecules are free to move sideways within a lipid bilayer.

P-type pump

Structurally and functionally related multipass trans- membrane proteins. They are called "P-type" because they phosphorylate themselves during the pumping cycle. This class includes many of the ion pumps that are responsible for setting up and maintaining gradients of Na+, K+, H+, and Ca2+ across cell membranes.

F-type ATP synthases

Structurally related to V-type pumps, often called ATP synthases because they normally work in reverse: instead of using ATP hydrolysis to drive H+ transport, they use the H+ gradient across the membrane to drive the synthesis of ATP from ADP and phosphate.

protein translocators in the mitochondrial membranes

TOM, SAM, OXA, TIM, TIM23, TIM22

confocal fluorescence microscope

The confocal microscope is generally used with fluorescence optics, but instead of illuminating the whole specimen at once, in the usual way, the optical system at any instant focuses a spot of light onto a single point at a specific depth in the specimen. This requires a source of pinpoint illumination that is usually supplied by a laser whose light has been passed through a pinhole. The fluorescence emitted from the illuminated material is collected at a suitable light detector and used to generate an image. A pinhole aperture is placed in front of the detector, at a position that is confocal with the illuminating pinhole—that is, precisely where the rays emitted from the illuminated point in the specimen come to a focus. Thus, the light from this point in the specimen converges on this aperture and enters the detector.

standard mass spectrometry (MS)

The first is the ion source, which transforms tiny amounts of a peptide sample into a gas containing individual charged peptide molecules. These ions are accelerated by an electric field into the second compo- nent, the mass analyzer, where electric or magnetic fields are used to separate the ions on the basis of their mass-to-charge ratios. Finally, the separated ions collide with a detector, which generates a mass spectrum containing a series of peaks rep- resenting the masses of the molecules in the sample.

different kinds of stimuli that open ion channels

The main types of stimuli that are known to cause ion channels to open are a change in the voltage across the membrane (voltage-gated channels), a mechanical stress (mechanically gated channels), or the binding of a ligand (ligand-gated channels). The ligand can be either an extracellular mediator—specifically, a neurotransmitter (transmitter-gated channels)—or an intracellular mediator such as an ion (ion-gated channels) or a nucleotide (nucleotide-gated channels).

fluorescence microscopy

The maximum excitation and emission RFP wavelengths of several commonly used fluorescent probes are shown in relation to the corresponding colors of the spectrum. The photon emitted by a fluorescent molecule is necessarily of lower energy (longer wavelength) than the absorbed photon and this accounts for the difference between the excitation and emission peaks. CFP, GFP, YFP, and RFP are cyan, green, yellow, and red fluorescent proteins, respectively. DAPI is widely used as a general fluorescent DNA probe, which absorbs ultraviolet light and fluoresces bright blue. FITC is an abbreviation for fluorescein isothiocyanate, a widely used derivative of fluorescein, which fluoresces bright green. The other probes are all commonly used to fluorescently label antibodies and other proteins.

major organelles

The nucleus contains the genome (aside from mitochondrial and chloroplast DNA), and it is the principal site of DNA and RNA synthesis. The surrounding cytoplasm consists of the cytosol and the cytoplasmic organelles suspended in it. The cytosol constitutes a little more than half the total volume of the cell, and it is the main site of protein synthesis and degradation. It also per- forms most of the cell's intermediary metabolism—that is, the many reactions that degrade some small molecules and synthesize others to provide the building blocks for macromolecules. About half the total area of membrane in a eukaryotic cell encloses the labyrinthine spaces of the endoplasmic reticulum (ER). The rough ER has many ribosomes bound to its cytosolic surface. Ribosomes are organelles that are not membrane-enclosed; they synthesize both soluble and integral membrane proteins, most of which are destined either for secretion to the cell exterior or for other organelles. We shall see that, whereas proteins are transported into other membrane-enclosed organelles only after their synthesis is complete, they are transported into the ER as they are synthesized. This explains why the ER membrane is unique in having ribosomes tethered to it. The ER also produces most of the lipid for the rest of the cell and functions as a store for Ca2+ ions. Regions of the ER that lack bound ribosomes are called smooth ER. The ER sends many of its proteins and lipids to the Golgi apparatus, which often consists of organized stacks of disc- like compartments called Golgi cisternae. The Golgi apparatus receives lipids and proteins from the ER and dispatches them to various destinations, usually covalently modifying them en route. Mitochondria and chloroplasts generate most of the ATP that cells use to drive reactions requiring an input of free energy; chloroplasts are a specialized version of plastids (present in plants, algae, and some protozoa), which can also have other functions, such as the storage of food or pigment molecules. Lysosomes contain digestive enzymes that degrade defunct intracellular organelles, as well as macromolecules and particles taken in from outside the cell by endocytosis. On the way to lysosomes, endocytosed material must first pass through a series of organelles called endosomes. Finally, peroxisomes are small vesicular compartments that contain enzymes used in various oxidative reactions.

scramblase in the plasma membrane

The plasma membrane also contains a scramblase but, in contrast to the ER scramblase, which is always active, the plasma membrane enzyme is regulated and only activated in some situations, such as in apoptosis and in activated platelets, where it acts to abolish the lipid bilayer asymmetry; the resulting exposure of phosphatidylserine on the surface of apoptotic cells serves as a signal for phagocytic cells to ingest and degrade the dead cell.

stability of sealed compartment formed by phospholipid bilayer

The same forces that drive phospholipids to form bilayers also provide a self-sealing property. A small tear in the bilayer creates a free edge with water; because this is energetically unfavorable, the lipids tend to rearrange spontaneously to eliminate the free edge. (In eukaryotic plasma membranes, the fusion of intracellular vesicles repairs larger tears.) The prohibition of free edges has a profound consequence: the only way for a bilayer to avoid having edges is by closing in on itself and forming a sealed compartment.

fixed angled rotor

The sample is contained in tubes that are inserted into a ring of angled cylindrical holes in a metal rotor. Rapid rotation of the rotor generates enormous centrifugal forces, which cause particles in the sample to sediment against the bottom sides of the sample tubes. The vacuum reduces friction, preventing heating of the rotor and allowing the refrigeration system to maintain the sample at 4°C.

principal features of a light microscope and a transmission electron microscope (TEM)

The source of illumination is a filament or cathode that emits electrons at the top of a cylindrical column about 2 m high. Since electrons are scattered by collisions with air molecules, air must first be pumped out of the column to create a vacuum. The electrons are then accelerated from the filament by a nearby anode and allowed to pass through a tiny hole to form an electron beam that travels down the column. Magnetic coils placed at intervals along the column focus the electron beam, just as glass lenses focus the light in a light microscope. The specimen is put into the vacuum, through an airlock, into the path of the electron beam. As in light microscopy, the specimen is usually stained—in this case, with electron-dense material. Some of the electrons passing through the specimen are scattered by structures stained with the electron-dense material; the remainder are focused to form an image, in a manner analogous to the way an image is formed in a light microscope. The image can be observed on a phosphorescent screen or recorded with a high-resolution digital camera. Because the scattered electrons are lost from the beam, the dense regions of the specimen show up in the image as areas of reduced electron flux, which look dark.

ultracentrifugation

The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape.

purifying proteins using genetically engineered tags

Using standard genetic engineering techniques, a short peptide tag can be added to a protein of interest. If the tag is itself an antigenic determinant, or epitope, it can be targeted by an appropriate antibody, which can be used to purify the protein by immunoprecipitation or affinity chromatography.

phase contrast light microscope

Variations in density with the specimen are amplified to enhance contrast in unstained cells; this is especially useful for examining living, unpigmented cells.

Passage of water molecules through an aquaporin channel

Water permeation through aquaporins (water channels) is a passive process that follows the direction of osmotic pressure across the membrane.

steps in the folding of a multipass transmembrane protein

When a newly synthesized transmembrane α helix is released into the lipid bilayer, it is initially surrounded by lipid molecules. As the protein folds, contacts between the helices displace some of the lipid molecules surrounding the helices.

how do lipid molecules react in an aqueous environment?

When amphiphilic molecules are exposed to an aqueous environment, they behave as you would expect from the above discussion. They spontaneously aggregate to bury their hydrophobic tails in the interior, where they are shielded from the water, and they expose their hydrophilic heads to water. Depending on their shape, they can do this in either of two ways: they can form spherical micelles, with the tails inward, or they can form double-layered sheets, or bilayers, with the hydrophobic tails sandwiched between the hydrophilic head groups

transmitter-gated ion channels at chemical synapses

When an action potential arrives at the presynaptic site, the depolarization of the membrane opens voltage-gated Ca2+ channels that are clustered in the presynaptic membrane. Ca2+ influx triggers the release into the cleft of small signal molecules known as neurotransmitters, which are stored in membrane-enclosed synaptic vesicles and released by exocytosis. The neurotransmitter diffuses rapidly across the synaptic cleft and provokes an electrical change in the postsynaptic cell by binding to and opening transmitter-gated ion channels.

interference between light waves

When two light waves combine in phase, the amplitude of the resultant wave is larger and the brightness is increased. Two light waves that are out of phase cancel each other partly and produce a wave whose amplitude, and therefore brightness, is decreased.

galactocerebroside

a neutral glycolipid; the sugar that forms its head group is uncharged

mass spectrometry

a technique that separates particles according to their mass

flippases

belongs to the family of P-type pumps, located on the plasma membrane. They specifically recognize those phospholipids that contain free amino groups in their head groups (phosphatidylserine and phosphatidylethanolamine) and transfers them from the extracellular to the cytosolic leaflet, using the energy of ATP hydrolysis.

dark field light microscope

brightly illuminated specimens surrounded by dark field