Exam 3 cell bio

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What are the four major types of cells junctions and their subtypes? How do these junctions functions. What are they composed of, and what cytoskeletal filaments do they link to if any?

- Anchoring junctions: adherens junctions (cell-cell junctions connected to actin, composed most frequently of classical cadherins); desmosomes (cell-cell junctions connected to intermediate filaments involving specialized cadherins); hemidesmosomes (cell-matrix junctions linked to intermediate filaments, usually involving integrin binding to components of the ECM); actin-linked cell matrix adhesion a.k.a. focal adhesions (cell-matrix adhesion linked to actin filaments involving integrin binding to components of the ECM). - Occluding junctions or tight junction form between neighboring cells. They form seals to prevent the movement of fluid between the apical and basolateral extracellular regions, as well as forming fences to prevent the movement of proteins in the membrane between the apical and basolateral regions of the plasma membrane. They are composed of occludins and claudins. - Gap junctions allows the passage of small water soluble of molecules directly between cells. They are composed of connexins that form connexons (composed of 6 connexins). A connexon on one cell forms an intracellular channel with a connexon on a neighboring cell.

Actin capping proteins

- SEE IMAGE -uncapped population of filaments- growth at plus and minus end - capped population of filaments- growth at minus end only; higher critical concentration 'growth only occurs under when we are above the critical concentration of the minus end

Cell movement and actin

- myosin moving along the actin filament is critical for cells to be able to move -actin polymerization of plus end protrudes lamellipodium - the stress fibers running through the cell which allows for tension and as myosin moves along it causes contraction to help the cell move - R23 allows for branching which allows the actin to move - back end is the myosin acting with the actin filaments allowing for the movement and actin assembling at the front end of the cell - cells form contact with basal lamina PHOTO SHOWS PROCESS

Bulletpointed Myosin Steps

-1: At the end of the previous round of movement and the start of the next cycle, the myosin head lacks a bound ATP and it is attached to the actin filament in a very short-lived conformation known as the 'rigor conformation'. -2: ATP binding to the myosin head domain induces a small conformational shift in the actin-binding site that reduces its affinity for actin and causes the myosin head to release the actin filament. -3: ATP binding also causes a large conformational shift in the 'lever arm' of myosin that bends the myosin head into a position further along the filament. ATP is then hydrolysed, leaving the inorganic phosphate and ADP bound to myosin. -4: The myosin head makes weak contact with the actin filament and a slight conformational change occurs on myosin that promotes the release of the inorganic phosphate. -5: The release of inorganic phosphate reinforces the binding interaction between myosin and actin and subsequently triggers the 'power stroke'. The power stroke is the key force-generating step used by myosin motor proteins. Forces are generated on the actin filament as the myosin protein reverts back to its original conformation. -6: As myosin regains its original conformation, the ADP is released, but the myosin head remains tightly bound to the filament at a new position from where it started, thereby bringing the cycle back to the beginning.

Cytoskeletal Filaments

-3 types: All have the first two, only some have the last one -Microtubules: a. Intracellular transport b. Organelle positioning c. Chromosome Segregation - Actin filaments: a. Cell shape b. Locomotion c. Cell Division (cytokinesis) - Intermediate filaments: a. Mechanical strength (not shown) b. Lamins: give nucleus shape, and forms associations w/ DNA/chromatin

Clathrin

-A fibrous protein found on the intracellular side of the plasma membrane (also associated with the Golgi complex) that helps invaginate the membrane. Typically cel surface receptors are associated with clathrin-coated pits at the plasma membrane binding of the ligan to the receptor trigger invagination (example: cholesterol uptake via lipoprotein endocytosis). -Subunit composed of 6 polypeptides (3 large, 3 small) • Triskelions assemble into basketlike shell • Important for endocytosis

Dynamic instability:

-A property of actin filaments in the cytoskeleton, characterized by rapid shortening or lengthening of individual filaments. • Capping proteins bind to the plus end • Stabilize microtubules -Rapid growth ensues with the GTP-capped end. The accidental loss of this cap causes a catastrophe, followed by rapid shrinkage of the microtubule. The Microtubule is rescued when it regains it GTP cap, allowing it to rapidly grow again. -Chemically speaking, the GTP-tubulin dimer (subunit of microtubule) attaches to other subunits, forming a polymer called a straight protofilament. GTP Hydrolysis changes subunit conformation, and weakens the bonds in the polymer. This causes a curved protofilament, which depolymerizes into GDP-Tubulin dimers, which undergo a GDP-GTP exchange to form GTP tubulin dimers, restarting the cycle

more power stroke of dyenin

-ATP binds to dyenin, microtuble released -ATP Hydrolyzed to ADP, phosphate release induces powerstroke

Troponins and Tropomyosin regulate Contraction

-At rest Troponin I-T interferes with tropomyosin positioning, so myosin cannot interact with actin. -In presence of Ca2+, Troponin C causes - Troponin I to release actin. -Tropomyosin shifts so myosin can bind actin and contraction can proceed. - calcium interacting with troponin causing tropomyosin to shift

TOPOLOGY You are studying a protein that normally resides in the plasma membrane. The organization of this protein is depicted inFigure 4 where the gray boxes labeled A, B, and C represent transmembrane regions and the segments labeled 1, 2, 3,Figure 15.5 and 4 represent the portions of the protein between the transmembrane regions. If you also know that segments 2 and 4 contain N-linked oligosaccharides, draw the topology of the protein as it is inserted in the plasma membrane. Be sure to label the cytoplasmic and extracellular face of your membrane and the N-terminus and C-terminus of your protein.

-Because segments 2 and 4 have N-linked oligosaccharides, they must have been facing the lumen of the ER and therefore will be extracellular. The topology of the protein in the membrane will be as depicted below. -In photo, N-terminus is on the left, C-terminus is on the right

Coat Proteins for vesicular transport: • COPI • COPII • Clathrin

-COP-coated vesicles involved in transport of vesicles from ER to Golgi and within Golgi, and. clathrin coated vesicles that carry proteins from the Golgi to the cell surface or from the cell surface to endosomes. • COPI: COPI is a coatomer, a protein complex that coats vesicles transporting proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum (ER), where they were originally synthesized, and between Golgi compartments. • COPII: COPII is a coatomer, a type of vesicle coat protein that transports proteins from the rough endoplasmic reticulum to the Golgi apparatus. This process is termed anterograde transport, in contrast to the retrograde transport associated with the COPI protein. • Clathrin: Clathrin performs critical roles in shaping rounded vesicles in the cytoplasm for intracellular trafficking. Clathrin-coated vesicles (CCV) selectively sort cargo at the cell membrane, trans-Golgi network, and endosomal compartments for multiple membrane traffic pathways.

For the following ER resident proteins, predict if they are soluble or transmembrane proteins: Calreticulin and HMG-CoA reductase

-Calreticulin is a soluble protein that will localize to the ER lumen, due to the presence of the KDEL ER retention signal at its C-terminus. -HMG-CoA reductase will be an ER transmembrane protein due to the KKXX (x=any amino acid) ER retention signal at its Cterminus

Nuclear export

-Cargo protein with nuclear export signal -Ran-GTP needs to be bound to Ran-GTP and the cargo -Will move through the nuclear pore complex out to the cytosol -Ran-GAP catalyzes GDP the hydrolysis of GDP to GTP -Dissociation

Different Coats for Vesicle Trafficking

-Clathrin -COPI (COP1) -COPII (COP2)

Sarcomere: Contractile Unit

-During contraction, thick filaments walk toward plus end of thin filaments. -Contraction requires ATPase activity of myosin and Ca2+ pumps - Plus ends attached to Z disc - myosin walks towards the plus end of the actin filament during contraction - the result is the two Z discs are brought closer together - the dark band is the thick filament and the light band is the actin filaments - for this to happen we need calcium and ATP -LOOK AT PHOTO FOR MORE

-ER is right next to the nucleus, as such, the lumen of the nuclear envelope is continuous with the ER lumen ER has 2 parts: a. rough ER: w/ ribosomes, site of protein synthesis. This is where post-translational modifications of proteins occurs. The formation of disulfide bridges also occurs here. i. Two different kinds of Ribosomes: those synthesized in the ReR (secreted into extracellular env, or becoming integral proteins in cell membrane, or in ReR, Golgi, or lysosomes) and cytoplasm (go to nucleus, peroxisomes, cytoplasm or mitochondria). b. Smooth: no ribosomes. Responsible for the synthesis of lipids, including those that will be in the cell membrane and those secreted from the cell (like steroid hormones). Metabolizes carbs, and aids in detox of drugs and other toxins.

ER resident proteins

-ER: responsible for quality control. It folds proteins-once proteins are folded, there are many places the protein could go. however, it goes through the Golgi. Afterwards, it can leave the cell, or go to another organelle -in the ER: proteins going to the Golgi, and those responsible for folding the incoming proteins. These are ER resident proteins -ER resident proteins never go to the Golgi -They have a unique tag called the KDEL sequence: -Lys-Asp-Glu-Leu (Lysine, Aspartine, Glutamine, leucine) . This addition is necessary and sufficient to keep the ER Res in the ER. -If the KDEL sequence was put on a protein that was meant to secrete out, it causes the protein to stay in the ER -If an ER res protein is mispacked, the secretory protein proceeds to the trans golgi membrane, But there are KDEL receptors inside of the trans-golgi network. These receptors can actually direct Cop1 assembly, which would send the vesicle and ER protein back to the ER lumen

GTP Hydrolysis by Ran Important for Export

-Exportin: nuclear export receptor -RanGAP (GTPase Activating Protein) -RanGEF (Guanine Exchange Factor) -cargo goes through nuclear export or is delivered to the cytosol

Microtubule Structure

-GTP/GDP-Hydrolysis affects microtubule dynamics -protofilament: plus end is more dynamic. Minus end is polar. -1 microtubule has 13 protofilaments -Tubulin heterodimer= microtubule subunit -PHOTO SHOWS MORE

Formation of Microtubule Bundles

-HALP -Map2 has one microtubule bind at one end and another on the other -tau has one microtubule on one end and another on the other end -Here, we demonstrate that under conditions of molecular crowding, tau forms liquid-like drops. Tubulin partitions into these drops, where it nucleates and drives the formation of microtubule bundles. These bundles deform the drops and remain enclosed by diffusible tau molecules, exhibiting a liquid-like behavior

Mouse L-cells are useful for studying the adhesive properties of cadherins, as they do not normally express cadherins. L-cells can be transfected with vectors expressing different cadherins to see how populations of cells behave. If two populations of cells are mixed that express different levels of the same cadherin, they form a ball with the highexpressing population on the inside. Why do you think they form a single ball with high-expressing cells on the inside?

-In the absence of calcium, cadherins cant form cel-cell anchoring junctions. Cells wouldn't migrate together in terms of development of stress. -Different types of cadherin in different places -If we took two populations of cadherins, the seperate populations would bind to themselves, since cadherin is homophyllic -can be sorted by levels of concentration: high expressing cad cells on inside, lower expressing on outside because the high expressing cadherins would join the ball first, bc there's more cadherins to associate with. This allows low expressing cells to only go on the outside, because the increased number of noncovalent bonds makes this more energetically favorable

Pathways to lysosome

-Intake of bacteria: phagocytosis. Take up of large particles or microorganisms. Results in a phagosome, which goes to the lysosome -Endocytosis for small particles. Take up of molecules from extracellular fluid. This results in an early endosome, which develops into a late endosome, which goes to the lysosome. -Disposal of cell parts requires autophagy, which results in an autophagosome, which ends up in the lysosome. -Nobel Prize in 2016:Yoshinori Ohsumi for Autophagy

Intermediate Filament Functions

-Maintain cell shape -Anchor the nucleus and organelles -Form the nuclear lamina -Types: a. Cytoskeletal 1. Keratin Filaments: Epithelial cells (Skin) 2. Vimentin/ Vimentin-related filaments: in connective tissue, muscle tissue and glial cells 3. Neurofilaments: In nerve cells b. Nuclear--> nuclear lamins in all animal cells

Microtubule Stability

-Microtubule Stabilization: Addition of MAP. The frequency of catastrophes on the GTP cap (the plus end of the microtubule) are suppressed, and/or the growth rates are enhanced. Results in longer, less dynamic microtubules -Microtubule Destabilization: Addition of catastrophe factor kinesin 13 causes the frequency of catastrophes on the GTP cap (the plus end of the microtubule) to increase, resulting in shorter, more dynamic microtubules

Mitochondrial import

-Mitochondrial proteins remain unfolded -TOM/TIM usually work together -Protein gets bound to receptor → receptor passes the protein to TOM complex → TOM complex passes the protein to TIM 23 → TIM 23 passes the protein to the matrix → Signal sequences are at the end and are cleaved off 1. Kicks off Hsp70. Requires ATP. Move across the outer mitochondrial membrane 2. Difference in charge provides energy to begin to pull the protein into the matrix. Inner mitochondrial membrane 3.Hsp70 proteins bind and associate to the protein. Pulls protein into the matrix. Requires ATP

Power Stroke of Myosin II: Part I

-Multiple myosin II molecules generate force in skeletal muscle through a power stroke mechanism fuelled by the energy released from ATP hydrolysis. The power stroke occurs at the release of phosphate from the myosin molecule after the ATP hydrolysis while myosin is tightly bound to actin. -PHOTO INCLUDES FIRST 3 STEPS: ATTACHED, RELEASED, COCKED

in multi-pass transmembrane proteins, the odd-numbered trans-membrane segments (counting from the N-terminus) act as start-transfer signals and the even-numbered segments act as stop-transfer signals

-Multiple signal sequences -Various starts determine orientation -Needs a protein that spans the ER membrane -N-terminus: start transfer sequence -Second signal sequence: stop transfer sequence -N-terminus ends up inside -C-terminus ends up outside

If you replace the normal Ran protein in the cell with a Ran protein that has an amino acid change locking Ran in a GDP-bound state (cannot exchange for GTP), how will this affect nuclear import and export?

-No import cargo released, no export cargo bound. -This is because for import, you need Ran-GTP to release cargo. For export, you need Ran-GTP to bind to the cargo

SNARE proteins

-Promote fusion of vesicles at the correct target membranes -Molecular Control of SNARE Complex Assembly SNARE complex assembly forces the two membranes to fuse in close proximity of each other. -In order to control when and where synaptic vesicle release occurs, it is critical to restrict and regulate SNARE complex assembly and disassembly. Mechanisms have been identified by which SNARE complexes are organized, and several classes of proteins have emerged as controllers of SNARE complex assembly: Two fundamental processes have been described: Munc18 proteins operate as clasps that control the assembly of four helix bundles, whereas complexin contains an α-helical domain that incorporates into SNARE complexes.

Nuclear import

-Protein beings with a nuclear import receptor -Protein gets passed through nuclear pore complex by binding to fibrils -Once cargo and receptor reach the nucleus, Ran-GTP binds to the receptor and causes the cargo to get released into the nucleus -Receptor with Ran-GTP moves from the nucleus to the cytosol -Ran-GTP is hydrolyzed into Ran-GDP

Rab and Rab Effectors- vesicular transport

-Rab proteins are small guanosine triphosphatases which regulate protein transport along the endocytic and exocytic pathways in all cell types. Rabs participate in vesicle budding, membrane fusion, and interactions with the cytoskeleton. Rab proteins are important for neuronal function because alterations in Rab protein regulation can lead to mental retardation in humans. In neurons, rab11 has emerged as a mediator of vesicle transport by coupling to myosin Vb to mediate GluR1 trafficking. Similarly, Rab8, which has an exclusive somatodendritic distribution, is involved in transport of proteins to the dendritic surface in neurons. In addition, Rab8 associates with exocyst in the recycling endosome and participates in targeting and delivery of AMPA receptors to the postsynaptic membrane. -Rab proteins constitute the largest branch of the Ras GTPase superfamily. Rabs use the guanine nucleotide-dependent switch mechanism common to the superfamily to regulate each of the four major steps in membrane traffic: vesicle budding, vesicle delivery, vesicle tethering, and fusion of the vesicle membrane with that of the target compartment. These different tasks are carried out by a diverse collection of effector molecules that bind to specific Rabs in their GTP-bound state. Recent advances have not only greatly extended the number of known Rab effectors, but have also begun to define the mechanisms underlying their distinct functions. By binding to the guanine nucleotide exchange proteins that activate the Rabs certain effectors act to establish positive feedback loops that help to define and maintain tightly localized domains of activated Rab proteins, which then serve to recruit other effector molecules. Additionally, Rab cascades and Rab conversions appear to confer directionality to membrane traffic and couple each stage of traffic with the next along the pathway.

Sorting in the trans Golgi network (end of Golgi)

-Secretory and membrane proteins have 3 different paths they could follow out of the trans-golgi 1. signal mediated diversion to lysosomes: utilizes mannose 6-phosphate receptor. 2. signal mediated diversion to secretory vesicles for regulated secretion 3.Constitutive secretory pathway. This is the default path

Ca2+ Induced Muscle Contraction

-Signal from nerve triggers action potential in muscle cell that is spread by T tubules to the myofibrils and SR. -Results in stimulation of voltage gated channel, which allows Ca2+ into cytosol. -Stimulates Ca2+ release channel so more Ca2+ released from SR and initiates contraction. -Sarcoplasmic reticulum (SR) stores intracellular Ca2+ -LOOK AT PHOTO FOR MORE

What type of amino acids are common in the start and stop transfer signal sequences in transmembrane proteins?

-Start and stop transfer signals for ER transport would be composed of primarily hydrophobic/nonpolar amino acids since these signals can function as transmembrane regions if they are located internally in the protein. -Look at amino acid table

Name the five translocases that are used to import proteins into the mitochondrion.

-TOM: The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins. It initially transports their signal sequences into the intermembrane space and helps to insert transmembrane proteins into the outer membrane -TIM23: The TIM23 complex then transports some of these proteins into the matrix space, while helping to insert transmembrane proteins into the inner membrane. -TIM22: The TIM22 complex mediates the insertion of a subclass of inner membrane proteins, including the carrier protein that transports ADP, ATP, and phosphate -OXA: A third protein translocator in the inner mitochondrial membrane, the OXA complex, mediates the insertion of inner membrane proteins that are synthesized within the mitochondria. It also helps to insert some proteins that are initially transported into the matrix by the TOM and TIM complexes. -SAM complex: Gets cargo from the inter membrane space. Moves B-barrel (porins) proteins into the outer mitochondrial membrane -Cytochrome oxidase: A signal sequence for mitochondrial protein import. 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

Pathways to the Lysosome Disposal of cell parts

-Take up of molecules from extracellular fluid -Take up of large particles or microorganisms -Disposal of cell parts -Nobel prize 2016

extracellular matrix (ECM)

-The meshwork surrounding animal cells, consisting of glycoproteins, polysaccharides, and proteoglycans synthesized and secreted by the cells. -important for absorbing mechanical stress (joints) • Organized network of proteins and polysaccharides (can interact with water) made by the cells with which it associates. • Regulates activity and shape of cells. -Fibroblasts create the proteins and sugars in ECM -basal lamina: organizes cells and maintains shape, right under epithelia

secretory pathway

-The movement of proteins produced by ribosomes attached to the ER, through a series of compartments and vesicles, via the Golgi apparatus, to secretory vesicles for export from the cell by exocytosis. -Rough ER -> Golgi -> secretory vesicles -> cell exterior -How does a protein know to go through a secretory pathway? They have a signal sequence that is detected during translation, causes protein to be pushed into ER

Myosin II

-The type of myosin that produces contraction by sliding actin filaments. • Composed of 2 heavy chains and 2 light chains a. conserved heavy chain w/ globular head domain b. light chains are light bc they must be specific to cargo • Heavy chain has globular head domain (conserved) and long tail (variable) that forms a coiled-coil with another tail. • Head binds and hydrolyzes ATP • Walks toward plus end of actin filament Myosin II forms Thick Filaments -heads on surface, tails within bundle -has a bare zone: On either side of the bare zone, parallel interactions extend the filament

The enzyme dihydrofolate reductase (DHFR) is normally located in the cytosol. If it is engineered to carry a mitochondrial targeting sequence at its N terminus, it is now imported into the mitochondria. If DHFR is first treated with methotrexate, methotrexate binds to the active site of DHFR, and the enzyme is no longer imported into the mitochondria. How do you think methotrexate interferes with mitochondrial import?

-This interferes with mitochondrial import because it targets the targeting sequence, which causes the protein to no longer be transported into the mitochondria -Methotrexate binding to the active site interferes with the mitochondrial targeting sequence, so the protein is no longer imported into the mitochondria.

unfolded protein response

-This response to an accumulation of unfolded/misfolded ER proteins results in an increased transcription of genes involved in retrotranslocation and cytosolic protein degradation. -Allows cells to expand the endoplasmic reticulum and produce more of the molecular machinery needed to restore proper protein folding and processing. -LOCATED IN LUMEN OF ER -There are three ER-resident transmembrane proteins orchestrating the UPR: the protein kinase R (PKR)-like ER kinase (PERK), activating transcription factor-6 (ATF6), and inositol requiring enzyme-1 (IRE1). Under normal resting conditions, these proteins are inactive because their luminal domains remain bound to GRP78. The accumulation of unfolded or misfolded proteins competes for GRP78 binding, thus liberating PERK, ATF6, and IRE1. This leads to the activation and downstream signaling of these three UPR effector proteins

Mitochondrial Translocators

-Translocase of Outer Membrane -β-barrels -Inner Membrane proteins made in mitochondrion and some others -Translocases of Inner Membrane

Actin Filament (Microfilament) Structure

-Two-stranded helix. -Flexible and easy to bend, easy to break. -Barbed end (1 actin molecule) is the plus end, and it is more dynamic -Pointed end is polar, and the minus end

Transport from the Trans Golgi to Lysosomes

-Vesicle from Golgi merges with lysosome • Lysosomes: a. Site of intracellular digestion b. Acidity maintained by H+ ATPase c. Enzymes (acid hydrolases) need low pH and cleavage to function -Lysosomal membrane proteins highly glycosylated for protection

Anchoring Junctions

-apical junctions -photo shows different types -cadherins: cell surface molecules

Assembly and Disassembly of Actin for Migration

-assembly at leading edge PHOTO: right= polymerization, left= depolymerization

Cadherins

-calcium-dependent glycoproteins that hold similar cells together • Ca2+ binds hinge region, prevents flexing, allows for adhesion • Interactions between many cadherins occur at junctions

Cell adhesion and cell sorting

-cells w similar origin associate • Cells with similar origins associate • Cells expressing similar levels of cadherin preferentially associate • Cells expressing the same type of cell adhesion molecules will preferentially associate.

Structure of the Basal Lamina

-grey= plasma membrane of cell connected to BL -green= integrins -laminin in blue

cilia and flagella

-hairlike structures that extend from the surface of the cell, where they assist in movement • Motility Structures • Composed of microtubules and dynein • 9 microtubles near periphery of the cell + 2 microtubules near the inside of the cell -For every pair of microtubles, one is moving, the other is cargo--> produces movement -bending of cilia allows for powerstroke to occur PHOTO

myosin thick filaments

-has numerous heads on the outside, and tails on the inside -muscles have muscle bundles, w muscle fibers. Within fibers, we have myofibrils made of sacromeres. Fibers have lots of nuclei

Integrins

-membrane proteins; they transmit signals between the ECM and cytoskeleton • Receptors for Extracellular Matrix Proteins • Can be activated from inside or outside the cell.

Transport from the ER through the Golgi

-newly synthesized proteins enter the biosynthetic- secretory pathway in the ER by crossing the ER membrane from the cytosol. These proteins pass through a series of compartments, where they are successively modified. -Transfer from one compartment to the next involves a delicate balance between forward and backward (retrieval) transport pathways. Some transport vesicles select cargo molecules and move them to the next compartment in the pathway, others retrieve escaped proteins and return them to a previous compartment where they normally function. The pathway from the ER to the cell surface involves sorting steps, which continually select membrane and soluble lumenal proteins for packaging and transport—in vesicles or organelle fragments that bud from the ER and Golgi apparatus. -a large proportion of the carbohydrates that it makes are attached as oligosaccharide side chains to the many proteins and lipids that the ER sends to it. A subset of these oligosaccharide groups serve as tags to direct specific proteins into vesicles that then transport them to lysosomes. But most proteins and lipids, once they have acquired their appropriate oligosaccharides in the Golgi apparatus, are recognized in other ways for targeting into the transport vesicles going to other destinations. • Transport from ER to Golgi via COPII vesicles • Cargo proteins have exit signals to target them to vesicles • Proteins must be properly folded/assembled for transport

Extracellular Matrix Molecules

-numerous proteins and proteins with specialized sugars (Glycosaminoglycan)

Proteins Leave the ER in COPII-coated Transport Vesicles

-proteins that have entered the ER and are destined for the Golgi apparatus or beyond are first packaged into small COPII-coated transport vesicles. These transport vesicles bud from specialized regions of the ER called ER exit sites, whose membrane lacks bound ribosomes. -In most animal cells, ER exit sites seem to be randomly dispersed throughout the ER network. packaging into vesicles that leave the ER can also be a selective process. Some cargo proteins are actively recruited into such vesicles, where they become concentrated. It is thought that these cargo proteins display exit (transport) signals on their surface that are recognized by complementary receptor proteins that become trapped in the budding vesicle by interacting with components of the COPII coat (Figure 13-17). At a much lower rate, proteins without such exit signals can also get packaged in vesicles, so that even proteins that normally function in the ER (so-called ER resident proteins) slowly leak out of the ER. Similarly, secretory proteins that are made in high concentrations may leave the ER without the help of sorting receptors.

Golgi Apparatus

-stack of membranes in the cell that modifies, sorts and sends prots to proper destination, and packages proteins from the endoplasmic reticulum -Synthezises molecules that need to be secreted • Composed of Cisternae (membrane enclosed compartments • Located near the ER • Resident proteins are membrane bound • Site of covalent modification • Site of O-linked glycosylation (Ser/Thr) -proteins enter the cis face of the golgi -Then they go to the medial stack. -Then comes the trans stack (furthest from ER). A vesicle buds off, and it can take. To the Lysosomes or ER (secretory pathway) or to outside of the cell, or cell membrane

Regulators of Microtubule Formation

-stathmin: binds subunits (alpha beta tubulin dimers) and prevents assembly -kinesin 13: enhances catastrophic disassembly at plus end for dynamic instability -kantanin: severs microtubules -Microtubules: filament building and cross-linking -MAP (microtubule associated protein): stabilize microtubles by binding along the sides -XMAP215: stabilizes plus ends and accelerates assembly

Actin Arrays

-stress fiber: contractile bundle -cell cortex: gel-like network -filopodium: Tight parallel bundle

Lamanin

-trimers like collagen -bind to integrins

What is the role of the extracellular matrix (ECM)? What are its components? What receptors bind the molecules of the ECM?

. The ECM consists of proteins and polysaccharides and performs a number of roles. It regulates cell polarity, shape, metabolism, migration, survival, differentiation, proliferation, and protein organization of the plasma membrane. It also bears mechanical stress. Specifically, its components include proteins (collagen, laminin, fibronectin, proteoglycans (proteins bound to glycosaminoglycans), glycosaminoglycans like hyaluronan, and water. Integrins are the most common receptors for components of the ECM.

Dynein powerstroke bulletpoints

1. ATP binds to dynein, which releases microtubule 2. Dynein repositions relative to microtubule 3. ATP hydrolyzed to ADP and Pi 4. When Pi is released, dynein binds strongly and the powerstroke occurs. 5. ADP is released

Nuclear Export

1. Cargo will bind well to the export receptor (exportin) when the receptor is bound to Ran-GTP. 2. The complex consisting of cargo/exportin/Ran-GTP will move through the nuclear pore complex into the cytosol. 3. Ran-GAP will act on the GTP bound to Ran, hydrolyzing it to Ran-GDP and causing the whole complex to fall apart. 4. Thus, the cargo will be released into the cytosol.

Intracellular Compartments

1. Endosome: sorting of endocytosted material. They regulate trafficking of proteins and lipids among other subcellular compartments of the secretory and endocytic pathway, specifically the plasma membrane Golgi, trans-Golgi network (TGN), and vacuoles/lysosomes. 2. Lysosome: responsible for degradation of enzymes. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules. 3. Cytosol: The cytosol serves several functions within a cell. It is involved in signal transduction between the cell membrane and the nucleus and organelles. It transports metabolites from their production site to other parts of the cell. It is important for cytokinesis, when the cell divides in mitosis 4. Peroxisome: have enzymes for oxidation. sequester diverse oxidative reactions and play important roles in metabolism, reactive oxygen species detoxification, and signaling. Oxidative pathways housed in peroxisomes include fatty acid β-oxidation, which contributes to embryogenesis, seedling growth, and stomatal opening. 5: Free ribosomes: protein synthesis 6. plasma membrane: provides protection for a cell. It also provides a fixed environment inside the cell. And that membrane has several different functions. One is to transport nutrients into the cell and also to transport toxic substances out of the cell. 7. nucleus: genome- DNA/RNA synthesis 8. ER w/ membrane-bound polyribosomes: protein and lipid synthesis 9. Golgi: protein/lipid modification and sorting. Processes materials to be removed from the cell. Makes/secretes mucus and packages products into vesicles for transport 10. Mitochondrion: ATP synthesis. It generates most of the chemical energy needed to power the cell's biochemical reactions. Chemical energy produced by the mitochondria is stored in a small molecule called adenosine triphosphate (ATP).

Transport between Compartments 2

1. Gated Transport (Nuclear Pore Complex) - between the cytosol and nucleus 2. Transmembrane Transport (Translocators) - between the cytosol and mitochondria/plastids/peroxisomes/ER 3. Vesicular Transport (Membrane-enclosed) - between the ER and the Golgi as well as between the Golgi and endosomes/lysosome/secretory vesicles/ cell exterior -Membrane proteins transferred, with same domain always oriented to same domain- any proteins that are embedded will have the same orientation - so if it is cytosol it will face cytosl if it is in the noncytosolic it will face the noncytosolic membrane) - vesicles can bud and fuse to move soluble components from one lumen to another

Put the following steps used to transport proteins into mitochondria into the proper order. (Mitochondrial protein transport)

1. Mitochondrial protein is synthesised in the cytosol. 2. The receptor on the mitochondrial membrane binds the signal sequence 3. Protein is delivered to the translocation apparatus in the mitochondria 4. Protein is passed through the translocation apparatus 5. The signal sequence is removed and the protein folds inside the mitochondria

types of cell junctions

1. anchoring junctions: allow cells to interact w/ eachother and ECM 2. occluding junctions (tight junctions) 3. Gap junctions

Fates of Endocytosed Transmembrane Proteins

1. recycling into plasma membrane 2. degradation in lysosome 3. transcytosis: macromolecules are transported across the interior of a cell. Macromolecules are captured in vesicles on one side of the cell, drawn across the cell, and ejected on the other side.

Nuclear Import

1. the cargo would bind to the import receptor (importin). The cargo/receptor complex would move through the nuclear pore complex into the nucleus. 2. Once in the nucleus, Ran-GTP binds to importin, causing it to release the cargo. The Importin/Ran-GTP complex is exported back to the cytosol. 3. In the cytosol, the GTP is hydrolyzed to GDP with the help of Ran-GAP. When Ran is bound to GDP, it no longer binds to importin. Thus, importin is now available to bind more cargo to start another round of nuclear import. Ran-GDP may be transported back into the nucleus as well, by its own import receptor. 4. Once Ran-GDP is back in the nucleus, Ran-GEF will act on Ran-GTP, causing it to release GDP and bind a new molecule of GTP.

Steps of muscle contraction

1.ACh released from synaptic vesicles 2.Binding of ACh to motor end plate 3.Generation of electrical impulse in sarcolemma (depolarization) 4.Conduction of impulse along T-tubules 5.Release of Calcium ions by SR 6.Calcium binds to troponin 7. Exposure of myosin binding sites on actin 8. Cross-bridge formation and contraction OR 1. Neuron stimulates a muscle cell 2. Action potential triggers the opening of Ca2+ channels 3. Ca2+ is released from the sarcoplasmic reticulum 4. Troponin moves tropomyosin protein 5. myosin interacts with actin

During vertebrate development, a sheet of epithelial tissue invaginates to form the neural tube, a structure that eventually forms the spinal cord and brain. Mutations that interfere with the function of which proteins would be most likely to disrupt the epithelial sheet movement that drives this developmental process?Choose one: A. cadherins B. connexons C. occludins D. claudins

Which is true of ribosomes?Choose one: A common pool of ribosomes is used to synthesize both cytosolic proteins and proteins destined for the ER. Polyribosomes translate only cytosolic proteins. All ribosomes are attached to the ER when they begin synthesizing a protein. A special class of ribosomes attached to the ER membrane translates the proteins destined for that organelle. Polyribosomes translate only those proteins that have an ER signal sequence.

A common pool of ribosomes is used to synthesize both cytosolic proteins and proteins destined for the ER.

lamellipodium

A sheetlike extension, rich in actin filaments, on the leading edge of a motile cell or growth cone. Composed of a network of actin filaments

Many viruses enter cells through receptor-mediated endocytosis. Which of the following strategies could be affective in blocking entry of this class of viruses into cells and could be used to treat viral infections?Choose one or more: A.Block the function of adaptin. B.Block the actin filaments. C.Increase the activity of clathrin. D.Block the receptor with an antibody.

A,D. For a virus to enter a cell via receptor-mediated endocytosis, the virus needs to bind the receptor and then adaptin binds the receptor to recruit clathrin. Blocking either the receptor or adaptin would block receptor-mediated endocytosis and viral entry.

What proteins regulate assembly, disassembly and stability of actin filaments and microtubules? Fill in the table that follows.

Actin Filaments, Microtubules and Intermediate Filaments

Regulators of Actin Filament Formation

Actin Subunits 1. formin- nucleates assembly and remains associated with the growing plus end 2. thymosin- binds subunits prevents assembly 3. ARP complex- nucleates assembly to form a web and remains associated with the minus end 4. profilin - binds subunits speeds elongation Actin Filaments 1.Cofilin- binds ADP actin filament (less stable region of the filament) , accelerates disassembly 2. fimbrin and alpha actinin 3. gelsolin - servers filaments and binds to plus end; cuts then to speed up disassembly 4. filamin and (spectrin-plasma membrane-ERM) = filament bundling, cross linking and attachment to membranes 5. capping proteins - prevents assembly and dissembly at plus end ; binds to the plus end of the actin filament and stabilize the end so that no addition or loss take place at that end 6. tropomyosin- stabalizes filament proteins that allow for actin bundling (parrellel allingment) - filamin and fimbrin proteins that help the actin cytoskeleton remain associated with the plasma membrane = spectrin and ERM

Myosin Functions

Associated with actin, walks towards plus end • Contractile activity (muscle and nonmuscle cells) • Vesicle and Organelle Transport cell division •Cytokinesis • Cell migration • Protrusion of actin-rich structures on cell surface • Construction of microvilli

For both clinical and cosmetic reasons, plastic surgeons inject substances into connective tissue underlying the skin epidermis. This plumps up areas deficient in soft tissue, and is used to reduce surgical scars or wrinkles, for example. Which normal connective tissue components are good candidates to be injected as fillers in this type of procedure?Choose one or more: A.hemidesmosome B.collagen C.cadherin D.glycosaminoglycan

B,D

You are interested in identifying the adhesion protein that is important to hold cells together in the developing zebrafish embryo. Your friend has antibodies to a number of cell surface proteins for you to test. An antibody to the protein of interest should prevent cells from associating. What protein would you expect to hold cells together in the developing embryo? You remove Ca2+ from the medium you are raising your zebrafish embryos in. What do you expect to happen to the embryo?

Cadherins are the primary adhesion proteins that mediate cell-cell adhesion in anchoring junctions. As these proteins require Ca2+ for their function, removal of Ca2+ from the culture medium would lead to a failure of cadherins to be in the extended conformation needed for their interaction with each other. Therefore, it is very likely that the cells would not be properly held together in the early embryo and it would not survive.

The outer membrane of the nucleus is continuous with the membrane of which other organelle?Choose one: A. Golgi apparatus B. mitochondrion C. endosome D. endoplasmic reticulum E. peroxisome

Which of the following is inconsistent with the function of gap junctions? Choose one: A. They allow inorganic ions to move directly between attached cells. B. They allow an electrical and a metabolic coupling between attached cells. C. They can open or close as needed. D. They allow ATP-driven pumps to move substances between attached cells. E. They allow the cytoplasm of two adjacent cells to be continuous with each other.

Botulism is a potentially fatal foodborne disease caused by the bacterium Clostridium botulinum. C. botulinum produces different toxins, several of which are proteases that cleave neuronal SNARE proteins. What normal process is blocked by cleavage and inhibition of SNARE proteins? Choose one: A. docking of vesicles to target membranes B. budding of vesicles from the endoplasmic reticulum C. entry of proteins with ER signal sequences into the ER lumen D. fusion of vesicles with target membranes

D. SNAREs help mediate vesicle membrane fusion (see image). In the absence of vesicle fusion, vesicle-stored neurotransmitters cannot be released into synaptic clefts, leading to paralysis.

Which organelle sequesters Ca2+ inside muscle fibers?

D. sarcoplasmic reticulum

How do proteins with different nuclear localization signals get efficiently transported by a single nuclear pore complex?

Different proteins bind to different nuclear import receptors, however, these import receptor all associate with the amino acids that line the nuclear pore complex to move their cargo into the nucleus.

Dynamic instability results in microtubules that grow or shrink rapidly. You have a situation where a microtubule is in the shrinking phase. a. What would need to occur for the microtubule to start growing again? b. How would an increase in tubulin concentration affect the ability of the microtubule to grow or shrink? c. What would happen if only GDP (no GTP) was present in the solution? d. What would happen if the solution contained an analog of GTP that could not be hydrolyzed to GDP?

Dynamic instability results in microtubules that grow or shrink rapidly. You have a situation where a microtubule is in the shrinking phase. a. The cell would need a sufficient amount of tubulin dimers in their GTP bound to allow for growth. This would re-establish a GTP-cap on the end of the microtubule. b. More tubulin dimers would provide a greater number of subunits for addition, and allow the microtubule to grow more quickly. Thus, while one microtubule may be shrinking, the newly released subunits can be rapidly added to a different microtubule. One's loss is another's gain. c. Subunits would be present in the GDP form, favoring shrinkage of microtubules due to a less stable structure, until all microtubules are disassembled. d. Microtubules would be more stable and continue growing if the GTP could not be hydrolyzed to GDP. This would occur until all subunits were part of a microtubule.

Extracellular Ca2+ flows into the cell during a muscle contraction, binding troponin and allowing myosin to walk along actin filaments. True or False. Explain

False. The Ca2+ that flows into the cell from the extracellular space through the voltagegated calcium channels is not sufficient to initiate a muscle contraction, but it does bind to the Ca2+-Gated Ca2+ release channel that is in the membrane of the sarcoplasmic reticulum, releasing much more Ca2+ that can initiate a muscle contraction by binding to troponin C. This shifts tropomyosin out of the way so that myosin can walk along the actin filament

Why would the loss of filamin in cancer cells be bad news for the cancers cells and good news for the patient?

Filamin links actin filaments together, which is necessary for lamellipodia formation and cell migration. If cancer cells do not have filamen and do not form lamellipodia, they are less likely to migrate and metastasize to a new location in the body. If the cells cannot metastasize it is easier to eliminate the cancer

What does the cellular motility of sperm depend on?

Flagella, which uses microtubules and dynein

Functions and Locations of Junctions

Hemidesmosomes got their name, as scientists previously thought they were simply half of a desmosome. Why did they think this and why were they incorrect?

Hemidesmosomes would have only had one cell with cell surface proteins linked to intermediate filaments. Therefore, it would look like half of the normal desmosome structure which normally has cell surface proteins interacting between two cells and linked to intermediate filaments on the interior of each cell. However, hemidesmosomes actually use different types of proteins, namely the integrins in their formation, as compared to the cadherins that are part of the desmosomes. In addition, hemidesmosomes are linked to proteins in the ECM, rather than being linked to another cell. Thus, while there are some similarities that are also some very key differences between desmosomes and hemidesmosomes.

The three pathways of the unfolded protein response differ in importance in different cell types, enabling cells to tailor the response to their individual needs. You join a lab that studies the relative importance of the UPR in different cell types. Your advisor gives you a new cell culture and directs you to determine which of the three pathways is the most important for that cell type. You first treat the cells with a kinase inhibitor. Given the results in Figure A, which pathway(s) might be important in these cells? Choose one: a. IRE1 b. ATF6 c. IRE1 and PERK d. PERK e. IRE1 and ATF6

IRE1 and PERK. Both IRE1 and PERK are protein kinases and would be inhibited by a kinase inhibitor. They also could be involved in the response in this cell line.

In order to explore the role of TACC3 the authors overexpress TACC3 by injecting embryos with TACC3 mRNA in Figure 1. What effect does increased TACC3 have on axon outgrowth velocity and retraction rate? (1 pt)

Increased TACC3 results in reduced axon outgrowth velocity and decreased retraction rate, resulting in an overall increase in axonal length.

Why do you think there may not be any known motor proteins that move along intermediate filaments?

Intermediate filaments lack polarity, so it would be difficult to have a motor protein that moved in a specific direction.

Authors also knockdown TACC3 using morpholinos in Figure 1. What effect does knockdown of TACC3 have on axon outgrowth velocity and retraction rate? How does this result compare to the effects of inhibiting TACC3 with KHS-101? (2 pts)

Knockdown of TACC3 reduced axon outgrowth velocity and increased retraction rate. Treatment with KHS-101 results in a similar effect, specifically reduced growth velocity and immediate axon retraction.

Formation of Microtubule Bundles p2

LOOK AT PHOTO

When a muscle is stimulated to contract, myosin heads walk along actin filaments in repeated cycles of attachment and detachment. Which of the following represents a correct description of the events in this cycle?

Myosin attaches to actin; ATP binding reduces the affinity of myosin for actin; myosin is "cocked" as its head is displaced along the actin filament; the power stroke puts myosin in a "rigor" configuration.

In figure 2, the authors examine the effects of the drug nocodazole. How does nocodazole affect microtubules? What was the effect of exposing the explants to the drug nocodazole in Figure 2? How were these effects altered upon overexpression of TACC3? (3 pts)

Nocodazole is a microtubule depolymerizing drug. Exposure to nocodazole resulted in reduced axon growth velocity, microtubule growth length, and lifetime microtubule length. Overexpression of TACC3 increased growth velocity and growth length in nocodazole treated samples, but did not significantly increase lifetime microtubule length.

Testing Signal Sequences

Normal signal sequences: ER protein w ER signal sequence relocated signal sequences: cytosolic protein w ER signal sequence

Cofilin preferentially promotes the disassembly of older actin filaments. What distinguishes older actin filaments from newer actin filaments?

Older actin filaments are more likely to have subunits in the ADP, rather that the ATP-bound form. This allows the distinction between older and newer filaments.

Transmembrane Proteins with an Internal Signal Sequence 33

Orientation favors most positively charged amino acids in the cytosol.

Lipid Bilayer Synthesis

Phospholipid synthesis occurs in the cytosolic leaflet of the ER membrane. Translocators move lipids between leaflets of the membrane.

Why do cells need translocases in the outer mitochondrial membrane if they already have porins?

Porins are only large enough to transport ions and small molecules, but not large enough for most proteins. In addition, the TOM complex first helps porins enter the intermembrane space before porins are threaded into the membrane by the SAM complex.

GTP Ran and Nuclear Import

Ran-GAP and Ran-GEF • Regulators of Ran Function • Results in compartmentalization of Ran-GTP and Ran-GDP

GTPases Regulate the Cytoskeleton

Rho: at back end of cell, helps pull back end of cell forward. Helps actin-myosin contraction. Rac: at the front end of cell, when a cell finds kemoattractant, this activates it, and helps polymerize cell cortex, allowing lamellipodium to move. pREVENTS RHO MOVEMENT

Quick Assembly/Disassembly

SEE IMAGE Subunits held together noncovalently. • Subunits disassemble and assemble where needed due to easy diffusion of subunits. • Accessory proteins regulators of this process.

Nucleotide Hydrolysis and Treadmilling

SEE IMAGE • Minus end = less dynamic • Plus end = more dynamic • Critical concentration: subunit loss = subunit addition C>>Cc, subunits added C<<Cc, subunits lost -It occurs when one end of a filament grows in length while the other end shrinks resulting in a section of filament seemingly "moving" across a stratum or the cytosol. This is due to the constant removal of the protein subunits from these filaments at one end of the filament while protein subunits are constantly added at the other end -Elongating the actin filament occurs when free-actin (G-actin) bound to ATP associates with the filament. Under physiological conditions, it is easier for G-actin to associate at the positive end of the filament, and harder at the negative end. However, it is possible to elongate the filament at either end. Association of G-actin into F-actin is regulated by the critical concentration outlined below. Actin polymerization can further be regulated by profilin and cofilin. Cofilin functions by binding to ADP-actin on the negative end of the filament, destabilizing it, and inducing depolymerization. Profilin induces ATP binding to G-actin so that it can be incorporated onto the positive end of the filament. -eadmilling occurs when one end polymerizes while the other end disassembles - The critical concentration Cc is the concentration of either G-actin (actin) or the alpha,beta- tubulin complex (microtubules) at which the end will remain in an equilibrium state with no net growth or shrinkage

Treadmilling

SEE IMAGE • Minus end has higher Cc than plus end. • Subunits added to plus end, lost from minus end. • T form = +GTP or +ATP; D form= + GDP or +ADP • Most common in actin filaments.

Transport to intermembrane space and inner membrane

Single transmembrane domain and multiple transmembrane domains

Slit normally functions as a repellent in the context of the nervous system. The authors test the effect of adding Slit to the their explants in Figure 4. What effect is observed on growth cone collapse? Next, the authors look at the effect of Slit when TACC3 is bring overexpressed-how is growth cone collapse affected under these conditions? (2 pts)

Slit induces growth cone collapse. Overexpression of TACC3 results in less growth cone collapse and axon retraction compared to samples not overexpressing TACC3.

Describe what is known about dynein walking.

Steps in Dynein Walking: • ATP binding to dynein causes it to release its association with the microtubule and repositions relative to the microtubule. • ATP hydrolysis to ADP and P. • Release of the phosphate group allows for the strong association of dynein with the microtubule and the power stroke. • ADP is released as the power stroke occurs, resetting things for another step along the microtubule toward the minus end.

Describe the steps in kinesin walking.

Steps in Kinesin Walking (after just completing a step): • The lagging head hydrolyzes ATP to ADP and releases the phosphate, and the neck linker unzippers from the head domain. The leading head releases ADP and binds to ATP. Neck linker zippers on to the new leading head. • Lagging head is pulled forward to the new leading head. • Kinesin continues walking toward the plus end of microtubules.

Describe the steps in myosin walking.

Steps in Myosin Walking (after just completing a step): • Binding of ATP causes myosin to release the actin filament and repositions relative to actin filament. • Hydrolysis of ATP to ADP and Pi and mysoin loosely associates with the actin filament. • Release of Pi allows for strong binding of myosin to the actin filament, and the powerstroke. • ADP dissociates from myosin during the powerstroke.

In the Background section, authors discuss their previous work on TACC3. What type of protein had the authors shown that TACC3 is and what was its role in the nervous system? (1 pt)

TACC3 is a +TIP protein that was previously demonstrated to regulate microtubule dynamics in the context of neuron axons.

What question are the authors attempting to answer in this paper? (1 pt)

The authors are interested in how TACC3 regulates axon outgrowth and if it has a role in axon guidance.

Next authors aim to identify the domain(s) of TACC3 that are important for axon outgrowth in Figure 1. What domain do they determine is required for this function? What have previous studies revealed about the importance of this domain? (2 pts)

The authors find that the TACC domain is the most important domain for regulation of axon outgrowth and overall axon length. Previous studies had demonstrated that the TACC domain is important for interaction with the microtubule polymerase (XMAP215) as well as for centrosome localization

ERGIC53

The exit signals that direct proteins out of the ER for transport to the Golgi and beyond are mostly not understood. There is one exception, however. The ERGIC53 protein seems to serve as a receptor for packaging some secretory proteins into COPII-coated vesicles. Its role in protein transport was identified because humans who lack it owing to an inherited mutation have lowered serum levels of two secreted blood-clotting factors (Factor V and Factor VIII) and therefore bleed excessively. The ERGIC53 protein is a lectin that binds mannose and is thought to recognize this sugar on Factor V and Factor VIII proteins, thereby packaging the proteins into transport vesicles in the ER.

What is the role of the unfolded protein response? Where does it function? What molecules are involved? By what mechanisms do these molecules function?

The function of the unfolded protein response is the increase the production of chaperone proteins in the ER lumen to assist in protein folding. There are three different ER membrane proteins that mediate the unfolded protein response: IRE1, PERK, and ATF6. Each of these mechanisms is activated by the accumulation of misfolded proteins. When ATF6 is activated, it is transported to the Golgi, where it is cleaved to release a portion of the protein that will migrate to the nucleus and function as a gene regulatory protein, meaning it will regulate transcription of the genes encoding ER chaperones. When PERK is active it is phosphorylated and it phosphorylates and inactivates a translation initiation factor, halting most protein translation to decrease the load of misfolded proteins. At the same time, we see translation of a gene regulatory protein that will increase transcription of genes encoding ER chaperones. In the case of IRE1, misfolded proteins activate IRE1, causing it to dimerize and phosphorylate. IRE1 will then splice an mRNA transcript encoding a gene regulatory protein that will promote the transcription of chaperone-encoding genes. We can see that for each of the mechanisms, they have a similar effect, to increase the production of chaperones and thereby decrease the number of misfolded proteins.

Glycosaminoglycan polysaccharide chains are highly negatively charged. How do you think this helps to establish a gel-like environment around the cell? Would this still occur of the chains were uncharged?

The negative charges allow glycosaminoglycans to form electrostatic interactions with water, creating a gel-like environment. If the chains were not charged, they would not interact with water well and the environment would be very different.

You have a protein with one N-terminal signal sequence, two stop transfer sequences and one start transfer sequence that you know if an ER membrane protein. How many transmembrane domains will this protein have?

The protein would have three transmembrane segments. The N-terminal signal sequence would be cleaved off. This would have functioned to start transfer into the ER. Then the two stop transfer sequences and one start transfer sequence would each function as transmembrane domains.

In Figure 3C and D, the authors examine the relationship between TACC3 and XMAP215. What do the results in this figure suggest about their relationship? (1 pt)

The results suggest that TACC3 and XMAP215 work together to regulate axon outgrowth.

Three separate pathways make up the unfolded protein response in the ER. Sort the following characteristics of the unfolded protein responses into the correct pathway. These are for IRE1, ATF6 and PERK.

These three different pathways use distinct mechanisms to activate transcription of genes to increase the protein-folding capacity of the ER.

You are interested in studying the role of different cytoskeletal filaments in cell motility, so you expose them to drugs that affect each type. When exposed to cytochalasin B, which caps actin filaments, locomotion of the cells is halted. Exposure to colchicine, results in the depolymerization of microtubules, and cells cease to move and they begin to send out lamellipodia in all directions. Exposure of a drug that interferes with intermediate filaments has no affect on locomotion. What do these results tell you about the roles of these different cytoskeletal filaments in locomotion?

Treatment with cytochalasin B reveals that polymerization at the plus end of actin filaments is necessary for cell motility, as this drug caps actin filaments to prevent their growth. Exposure to colchicine results in the depolymerization of microtubules and a failure to send out lamellipodia in one direction of movement. This suggests that microtubules are important for directional movement. If the formation of lamellipodia is not directed, the cell is pulled in multiple directions and fails to undergo migration. As a drug interfering with intermediate filaments does not affect cell motility, these cytoskeletal filaments are not required for cell motility

In a centrosome, which structures serve as nucleation sites for the formation of microtubules?Choose one: A. γ-tubulin rings B. α- and β-tubulin monomers C. tubulin protofilaments D. αβ-tubulin dimers

Between which compartments do the following types of transport occur? a. Gated Transport b. Transmembrane Transport

a. Gated: Nucleus and cytosol, Allows small molecules to move freely, Large molecules need receptor proteins and Ran to move in or out. b. In and out of a cell. Examples would be uniports, symports etc. Look at photo for more examples

Clathrin coated vesicles bud from the plasma membrane when adaptor proteins, clathrin and dynamin are added. What would happen under each of the following conditions? a. No adaptor proteins b. No clathrin c. No dynamin

a. Without adaptor proteins, the vesicle coat proteins would not be added. b. Without clathrin, the vesicle would not mold and proteins would not be properly aggregated in the membrane for the process of endocytosis. c. Without dynamin, the vesicle would not pinch off.

An oligosaccharide is added to a protein in the ER. Where will the part of the protein with the sugar be oriented?Choose one: A. It could be located in the ER lumen or the cytosol, depending on the protein. B. In the ER lumen C. In the middle of the ER lipid bilayer. D. In the cytosol

Name the organelle that proteins can enter as they are being synthesized.Choose one: A. peroxisome B. ER C. mitochondrion D. chloroplast E. nucleus

What type of translocase would perform the final step of moving a multi-pass transmembrane protein made in the cytosol into the inner mitochondrial membrane?Choose one: A. TOM B. TIM22 C. SAM D. OXA E. TIM23

Which is true of the constitutive exocytosis pathway?Choose one: a. It packages proteins in secretory vesicles that accumulate near the plasma membrane. b.It operates continually in all eukaryotic cells.It packages proteins that carry a signal sequence that marks them for secretion. c. It replaces the regulated exocytosis pathway in cells specialized for secretion. d. It packages proteins that form aggregates in the conditions present in the trans Golgi.

Which proteins play a central role in the fusion of a vesicle with a target membrane?Choose one: a. tethering proteins b. SNAREs c. clathrin d. adaptin e. Rab proteins

You complete a further experiment by treating your cells with an RNAse inhibitor and get the results shown in Figure B. Given the results of Figure A and Figure B, what pathway(s) is/are important for this cell line? Choose one: a. IRE1 and PERK b. IRE1 c. ATF6 d. PERK e. IRE1 and ATF6

b. IRE1 has kinase and RNAse domains. Inhibition of either domain blocks pathway activation and will lower the unfolded protein response in this cell line.

Insulin is synthesized in the form of a precursor protein that requires cleavage of two different peptide segments before the mature protein is secreted from β cells in the pancreas. The first peptide is removed when the protein enters the lumen of the ER. To find out when the second cleavage event takes place, investigators prepare a pair of antibodies: one recognizes the pro-insulin precursor, the other the mature insulin protein. They tag the antibody that binds to the precursor protein with a red fluorescent marker; the antibody that binds to mature insulin is tagged with a green fluorescent marker. When both markers are present, the sample fluoresces yellow. The investigators then incubate an isolated β cell with both antibodies at the same time and monitor the fluorescence in its various membrane-bound compartments. The data are shown in the table below. Based on these observations, where is the second peptide removed from the pro-insulin precursor protein? Choose one: a.mature secretory vesicles GREEN b. immature secretory vesicles YELLOW c. lysosomes NONE d. Golgi apparatus RED e. ER- RED

b. The appearance of yellow fluorescence in the immature secretory vesicles indicates that both the precursor protein and mature insulin are present, which suggests that the cleavage is taking place here.

Nuclear import is driven by the hydrolysis of GTP, which is triggered by an accessory protein called Ran-GAP (GTPase-activating protein). Which is true of this process?Choose one: a. Ran-GDP displaces proteins from nuclear import receptors inside the nucleus. b. Ran-GAP is present exclusively in the nucleus. c.Nuclear receptors carry Ran-GTP from the nucleus to the cytosol. d. Nuclear import receptors have the ability to catalyze hydrolysis of GTP. e. Ran-GTP is present in high concentrations in the cytosol.

Proteins entering the cis Golgi network can do which of the following?Choose one: a. They are sorted according to whether they are destined for lysosomes or for the cell surface. b. They can either move backward to the trans Golgi network or be sent forward to the plasma membrane. c. They can either move onward through the Golgi stack or be returned to the ER. d. They are either sent to the nucleus or to the plasma membrane for secretion. e. They can be held in the cis Golgi while they are properly folded and assembled.

Which best describes a pathway that a protein might follow from synthesis to secretion?Choose one: a. Cytosol → ER → secretory vesicle → plasma membrane b. ER → Golgi apparatus → secretory vesicle → plasma membrane c. Cytosol → ER → transport vesicle → Golgi apparatus → transport vesicle → plasma membrane d. Cytosol → ER → Golgi apparatus → transport vesicle → endosome → secretory vesicle → plasma membrane e. ER → Golgi apparatus → transport vesicle → endosome → secretory vesicle → plasma membrane

In a classic experiment designed to study nuclear transport, investigators added a dye molecule to the subunits of a protein called nucleoplasmin, which is involved in chromatin assembly. They then injected the intact protein or combinations of its subunits into the cytosol of a frog oocyte or into its nucleus.The results of the experiment are shown in the diagram, where red indicates the location of the labeled protein Based on these results, which part of the nucleoplasmin protein bears a nuclear localization signal? Choose one a.both the head and the tail b.the head only c.the tail only d. neither the head nor the tail e.No conclusion about the nuclear localization signal can be drawn from the data.

c. Only the preparations that have at least one tail are able to enter the nucleus after being injected into the cytoplasm (column on the right).

Which of the following components of receptor-mediated endocytosis of LDL is incorrectly matched with its function?Choose one: A. clathrin: forms the coated vesicle B. adaptin: binds to the specific receptors and recruits clathrin C. lysosome: releases LDL from the receptor D. LDL receptors: form bridges between the LDL particle and adaptin

c. The lysosome is not where LDL is released from the receptor. The LDL is released from the receptor in the endosome before being transported to the lysosome for release of cholesterol.

Researchers studying yeast discovered that, for some mutants, when the temperature at which the cells are grown is elevated from 25ºC to 37ºC, their secretory pathway no longer functions and the cells grow dense with unsecreted protein.When these cells are examined microscopically, they can be divided into groups that vary in terms of where the unsecreted proteins accumulate. In some of the mutants, proteins accumulate in the ER; in others, the Golgi; in others, they accumulate in vesicles near the plasma membrane.What is the likely explanation for this difference in appearance?Choose one: a.Different temperature-sensitive mutations disrupt protein synthesis. b. Different temperature-sensitive mutations disrupt the integrity of cell membranes. c. Different temperature-sensitive mutations affect different stages of the transport process. d. The temperature-sensitive mutant proteins accumulate in different compartments. e. Different temperature-sensitive mutations promote an increase in protein synthesis.

c. This approach was used by one research group to identify at least 23 different genes required for the transport of proteins from their site of synthesis to their secretion at the cell surface.

Conformations of the Import Receptor

cargo loading/cargo unloading

Proteins that lack a sorting signal remain as permanent residents of which part of a eukaryotic cell?Choose one: ER cytosol nucleus Golgi apparatus proteasome

cytosol

An actin filament undergoing treadmilling at the leading edge of a lamellipodium can do what?Choose one: a. collapse and instantly disappear b. experience exponential growth c. add actin monomers to its minus end while losing them from its plus end d. remain the same size

If GTP hydrolysis occurs on a tubulin molecule at the plus end of a microtubule protofilament before another tubulin molecule is added, what typically happens?Choose one: A. The microtubule remains the same size. B. The microtubule polymerizes. C. The GDP is rapidly exchanged for a fresh molecule of GTP. D. The microtubule depolymerizes.

Investigators have engineered a gene that encodes a protein bearing an ER signal sequence followed by a nuclear localization signal. What would be the likely fate of that protein? Choose one: a. Because of its conflicting signals, the protein will be degraded in the cytosol. b. The protein will be recognized by a nuclear import receptor and escorted into the nucleus. c. Because of its conflicting signals, the protein will be sent to a lysosome for destruction. d.The protein will be recognized by an SRP and enter the ER. e. Because of its conflicting signals, the protein will remain in the cytosol.

What is the main function of intermediate filaments?Choose one: A. They enable muscle cells to contract. B. They provide tracks for guiding intracellular transport. C. They enable cells to crawl. D. They enable cells to withstand the mechanical stress that occurs when cells are stretched. E. They segregate chromosomes during cell division.

Which cellular compartment acts as the main sorting station for extracellular cargo molecules taken up by endocytosis?Choose one: a. transport vesicles b. clathrin-coated vesicles c. lysosomes d. endosomes e. Golgi apparatus

ATP is important for chaperone protein function. Why would protein import into mitochondria be disrupted if ATP were depleted from inside mitochondria?Choose one: A. The protein would be blocked from entering the translocation apparatus. B. The signal sequence would not be recognized on the mitochondrial protein. C. The translocation apparatus would be unable to function without ATP hydrolysis. D. The protein could slip back out of the mitochondria during transport.

d. Chaperones require ATP to help pull proteins into the mitochondrial matrix and prevent their exit back through the translocation channel.

What recognizes a signal sequence in a protein and directs a the protein, ribosome, and mRNA to the ER?Choose one: A. Protein Translocase B. Ran C. Rab D. Receptor in the ER membrane E. Signal Recognition Particle (SRP)

Ricin is one of the most powerful toxins known. The protein consists of two subunits: the A chain is an enzyme that inhibits translation and the B chain is a lectin that binds to carbohydrates on the cell surface. What is the most likely mechanism by which ricin enters the cell?Choose one: a. The A chain stimulates autophagy. b. The protein enters through pore complexes in the plasma membrane. c. The A chain binds to clathrin. d. The B chain interacts with SNAREs. e. The protein is internalized by endocytosis.

e. Ricin is taken up by endocytosis, most likely mediated by its binding to cell-surface receptors.

Exocytosis and Endocytosis

exocytosis: Delivery to membrane/extracellular space endocytosis: Internalization from the membrane/extracellular space

Power Stroke of Myosin II: Part II

force generating and attached

Assembly of Adherens Junctions

function: hold cells together so that they move together. Allow for the coordinated movement of cells

Transport between Compartments

gated transport: A gated channel protein is a transport protein that opens a "gate," allowing a molecule to pass through the membrane. Gated channels have a binding site that is specific for a given molecule or ion. A stimulus causes the "gate" to open or shut. EXAMPLE: transport through nuclear pores protein translocation: a process by which proteins move between cellular compartments. Short amino-acid sequences within a protein, known as signal peptides or signal sequences, can direct its localisation, although translocation also occurs in the absence of these signal sequences. EXAMPLE: proteins made in cytosol being brought to different cell compartments vesicular transport: a membrane protein that regulates or facilitates the movement of specific molecules across a vesicle's membrane. As a result, vesicular transporters govern the concentration of molecules within a vesicle. EXAMPLE: ER--> Endosome or Golgi--> out of cell or out of cell/ somewhere else in cell

To which type of junction: A) Gap, B) Tight or C) Anchoring, do the following statements apply? i) Seals adjacent membranes together to prevent movement of extracellular molecules. ii) Allows small molecules to move between the cytoplasm of adjacent cells. iii) Associated with filaments that can mediate changes in cell shape or impart mechanical strength.

i) B ii) A iii) C

Microtubule motor proteins

kinesin and dynein and dynactin PHOTO SHOWS LOCATIONS AND DIRECTIONS

Kinesins

microtubules, walks towards plus end • Composed of 2 heavy and 2 light chains • Primarily plus end directed microtubule motors • Head domain: motor that consumes ATP (conserved) • Tail domain binds cargo (variable) PHOTO SHOWS STRUCTURE

Proteins have to be unfolded to cross the membranes of which of these organelles?Choose one or more: mitochondria chloroplasts endoplasmic reticulum nucleus

mitochondria chloroplasts endoplasmic reticulum

Free vs. Membrane Bound Ribosomes

ribosomes are located in the cytosol. some are free and some are located on membrane bound organelles

Microvilli: Actin Bundles

see image for diagram

When a muscle is stimulated to contract, what does Ca2+ bind to, and what effect does that have?

troponin, which moves the tropomyosin that otherwise blocks the interaction of actin and myosin

Pinocytosis

• "Cell drinking" • Ingestion of fluid and small solutes

Phagocytosis

• "Cell eating" • Ingestion of large particles like microorganisms/dead cells by specialized cells. - ex: Phagocytosis of Red Blood Cell by Macrophage

Focal Adhesions

• Actin-linked cell matrix adhesions: attachments to ECM important for cell movement • Integrin binds ECM and is linked intracellularly to actin filaments. • Unstable, temporary structures

Assembly and Disassembly of the Coat

• Adaptor proteins: Bind clathrin coat to membrane and trap proteins in forming vesicle. Provide specificity.

Adherens Junctions allow Cellular Coordination

• Adhesion belt allow coordinated movement of epithelial cells as sheets.

Translocation of Soluble Proteins

• Binding of start transfer sequence (red) results in opening of the translocator and movement of protein into the ER lumen.

Profilin Regulates Actin Filament Formation

• Binds actin to prevent association with minus end, can only add to plus end. • Addition of actin to plus end results in conformation change and release of profilin.

trans-SNARE complex

• Brings membranes together for fusion • v-SNARE: on vesicle membrane, single polypeptide • t-SNARE: on target membrane, 2-3 polypeptides • Different SNAREs mediate fusion with different organelles

Desmosomes

• Cadherin-family adhesion proteins -made of cadherins • Association with intermediate filaments and provide mechanical strength

Capping proteins

• Capping proteins bind to the plus end • Stabilize microtubules -See image to understand how

Exocytosis in Mast Cells and Pancreatic Islet Cells

• Causes release of histamine in allergic response • Insulin release from pancreatic Beta-islet cells

Cellular Processes Regulated by the ECM

• Cell polarity • Cell metabolism • Protein organization in PM • Cell survival • Cell Proliferation • Cell Differentiation • Cell migration

Cadherin Superfamily

• Cell-cell adhesion • Adherens junctions: cadherin attaches to actin • Desmosomes: cadherin attaches to intermediate filaments. -either folded or extended conformations (in presence of CA 2+

Microtubule Organizing Center (MTOC)

• Centrosome: MTOC Microtubule Organizing Center in animal cells • Centrosome contains pair of centrioles, which ensure centrosome duplication during cell division. • Centrosomes organize mitotic spindle. -microtubules grow at at their plus ends from the γ-tubulin ring complexes of the centrosome (like little pores)

Protein Translocation

• Co- or Post-translational import into the ER in unfolded state -co-translational translocation -Post-translational translocation

Intermediate Filament Formation

• Coiled-coils (2 filaments) form tetramers (4 filaments). Structures of 8 tetramers • Strong, ropelike, easy to bend, hard to break. • Does not bind triphosphate. • No Polarity • Forms in cytoplasm of some cells.

Vesicular Transport

• Compartments have chemically distinct characteristics. • Combination of molecular markers on cytosolic surface give each compartment different address. • Vesicles deliver components to each compartment RED: Biosynthetic-secretory Pathway GREEN: Endocytic Pathway BLUE: Retrieval Pathways

Construction of Organelles

• Daughter cell inherits organelles from its mother during cell division. • Many organelles enlarge and are distributed to two daughter cells during division (ER, Golgi, Lysosome, Endosome, Peroxisome). • Inheritance of important translocators in the cell membranes of these organelles, so proteins are targeted to the correct organelle.

Gap Junctions

• Direct passageways between cells -allowed by connexons, made of connexins • Allows exchange of small molecules • Inserted into membrane via exocytosis

Modifications in the ER

• Disulfide bond formation • N-linked oligosaccharide chain added to target Asn of the polypeptide in the ER lumen.

Pinching Off and Uncoating of Vesicles

• Dynamin performs many functions: • Recruits proteins for pinching off.

Functions of Gap Junctions

• Electrical coupling for rapid spreading of action potentials. • Sharing of small molecules so cells coordinate their actions. • Switch between open and closed states

Receptor-mediated Endocytosis

• Endocytosis of macromolecules bound to receptors via clathrin-coated vesicles. • Example: Cholesterol taken up as a component of low-density lipoproteins.

Intermediate Filament and Disease

• Epidermolysis bullosa complex a. Linked to mutations in keratin b. Failure to form filaments, cannot withstand stress resulting in cell rupture • Amyotrophic lateral sclerosis (ALS) a. Accumulation of neurofilaments in the cell body and axons • Progeria a. Defects in nuclear lamins

Occluding (Tight) Junctions- prevent movement

• Epithelia line surfaces and cavities of the body • Apical side free; Basal side anchored to basal lamina • Tight junctions important for this cell polarity. • Encircle entire cell. a. Seals-prevents movement of nutrients and small molecules. b. Fences-prevent movement of membrane proteins

Movement of Cilia and Flagella

• Flagella-sperm and protozoa • Cilia-Paramecium and cells like those lining the respiratory tract and oviduct Attachment to B Microtubule Attachment to A Microtubule PICTURE DEPICTS PROCESS

Formins and Profilin

• Formin whiskers have multiple profilin binding sites. • Can increase rate of actin elongation. -reloading actin of formin whiskers can cause continues, rapid growth of actin filament at the plus end

Actin Filament Nucleation: Formins

• Formins form dimer that recruits two actin monomers. • Associate with plus end.

Types of Coated Vesicles

• Functions: a. Concentrates membrane proteins in specialized patches. b. Molds the vesicle • Coats discarded before membrane fusion. • Different coats for different compartments. TYPES OF COATED VESICLES

Smooth Endoplasmic Reticulum

• Functions: Hormone Synthesis, Detox in Liver, Intracellular Ca2+ Storage • Network of Tubules • No ribosomes • Vesicle Transport to Golgi

Rough Endoplasmic Reticulum

• Functions: Lipid and Protein Biosynthesis • Flattened Sacs • Membrane-bound ribosomes

Exocytosis

• Fusion of vesicles with the plasma membrane -CONSTITUTIVE SECRETION: All cells Continuous Specialized cells -REGULATED SECRETION: That secrete hormones, NTs, digestive enzymes -PHOTO SHOWS PROCESSES

GTP Binding Proteins as Molecular Switches

• GTP binding proteins switch functions based on GDP- or GTP-bound state. • GEF (guanine nucleotide exchange factor): causes release of GDP so GTP can bind. • GAP (GTPase activating protein): induces hydrolysis of GTP to GDP.

Weblike Networks

• Gel-like • Filamin links actin filaments • Important for lamellipodia formation • Loss of filamin in tumor cells makes them less invasive.

Proteoglycans

• Glycosaminoglycans linked to a protein core. • Create a hydrated "gel" that resists compression • Important in connective tissues • Bind and regulate activity of other secreted molecules examples: decorin, aggrecan, ribonuclease

Cadherins and cell-cell adhesion (embryos)

• Homophilic binding: binding between the same type of molecule • Heterophilic binding: binding between different types of molecules • Cadherins bind homophilically more frequently.

Hemidesmosomes

• Integrin anchors cells to the ECM and is coupled to intermediate filaments intracellularly. • Stable Structures= more stable connection to ECM -seen in skin

Cadherins link to the cytoskeleton

• Intracellular anchor proteins link cadherin to the cytoskeleton. • Connected to actin in adherens junctions • Connected to intermediate filaments in desmosomes -prefer to associate with similar molecules--> homophilic binding

Integrins p2

• Involved in cell-matrix and cell-cell adhesion. • Receptors for extracellular matrix proteins • 2 Subunits: α and β • Intracellular portion binds proteins that link receptor to the cytoskeleton.

Retrieval of ER resident proteins

• KDEL: ER retrieval signal on soluble proteins, binds receptor • KKXX: ER retrieval signal on membrane proteins, binds COPI coat Fusion via transSNARE complex

Power Stroke of Kinesin

• Lagging head in ATP state, zippers neck linker to throw other head forward to bind microtubule • Lagging head hydrolyzes ATP to ADP + Pi , Pi released, neck linker unzips and head pulled forward as front head releases ADP and binds ATP and neck linker zippers onto head to pull lagging head forward PHOTO IS VISUAL REPRESENTATION OF POWER STROKE

Assembly of Collagen outside the cell

• Made and secreted by fibroblasts (skin, tendons, connective tissues),osteoblasts (bone), and others -undergoes cleavage • Undergo exocytosis • Assembled extracellularly • Fibroblasts influence collagen organization -ECM also helps to organize collagen organization

Collagen

• Major component of extracellular matrix • Triple helix structure (3 collagen subunits in one molecule) • Form fibrils

Transport of Lysosomal Hydrolases

• Mannose 6-phosphate (M6P) added to lysosomal hydrolases • M6P receptor in trans Golgi recognizes M6P to target proteins to the endosomes.

Endocytosis

• Material taken into cell by invagination of the plasma membrane. -photo contains process

Mitochondrial Import

• Mitochondrial proteins remain unfolded during import • TOM/TIM usually work together for import -import receptors -translocation into matrix

Transcytosis

• Movement from one extracellular space to another. • Transcytosis (BREAST MILK) used for transfer of antibodies from mother's milk to baby.

Nucleation of microtubules

• Nucleation at the γ-tubulin ring complex (γ-TuRC), which is part of the Microtubule Organizing Center (MTOC) • Complex associates with the minus end

Actin Filament Nucleation: ARP complex

• Nucleation occurs at cell cortex (region just under plasma membrane) • ARPs: Actin-related proteins, associate with minus end • Involved in lamellipodia formation at leading edge for movement. -An inactive ARP complex contains Arp3, Arp2, and other proteins. An activating factor and actin monomers added allows for the formation of a nucleated actin filament, with the active ARP complex on the minus end of the filament.

Transport between the Nucleus and Cytosol

• Nucleus enclosed by nuclear envelope, consists of 2 membranes. • Inner: anchors chromatin and provides structural support • Outer: Continuous with the ER • Perinuclear space continuous with ER lumen. • Nuclear Pore Complexes used for transport in and out. • Post-translational Transport in the folded state -The nuclear envelope consists of an inner and an outer nuclear membrane. The outer membrane is continuous with the ER membrane, and the space between it and the inner membrane is continuous with the ER lumen. RNA molecules, which are made in the nucleus, and ribosomal subunits, which are assembled there, are exported to the cytosol, while all the proteins that function in the nucleus are synthesized in the cytosol and are then imported. The extensive traffic of materials between the nucleus and cytosol occurs through nuclear pore complexes, which provide a direct passageway across the nuclear envelope. Proteins containing nuclear localization signals are actively transported inward through the nuclear pore complexes, while RNA molecules and newly made ribosomal subunits contain nuclear export signals that direct their active transport outward through the pore complexes. Some proteins, including nuclear import and export receptors, continually shuttle between the cytosol and nucleus. -The GTPase Ran, provides directionality for nuclear transport. The transport of nuclear proteins and RNA molecules through the pore complexes can be regulated by denying these molecules access to the transport machinery. Because nuclear localization signals are not removed, nuclear proteins can be imported repeatedly, as is required each time that the nucleus reassembles after mitosis.

Structure of Tight Junctions

• Occludins and Claudins associate to form sealing strands • Multiple strands make up tight junction

Origins of the Compartments of the Secretory and Endocytic Pathways

• Pink areas are similar in environment to the exterior of the cell.

Structure of the Sarcomere

• Plus end of actin filaments anchored in Z disc by Cap Z protein (capping proteins that allow anchoring). This stabilizes the plus end, not allowing it to grow • Myosin walks toward plus end of actin filament -Photo includes locations of tropomodulin (stabilize overll actin filaments on minus end) , M line in photo -Mysosin= thick filament moves toward z disc -Actin= thin filament, shown as green helical structures in photo

Mitochondrial Porins

• Porins: β-barrels • Located in the outer membrane • Make membrane permeable to ions and small solutes (not proteins) • Inserted into membrane by SAM complex

Mitochondrial Protein Import

• Post-translational import in the unfolded state • Signal Sequence for matrix proteins: amphiphilic alpha helix for matrix proteins • + aa's on one side, hydrophobic on the other • Recognized by receptors

Motor Proteins

• Proteins that bind cytoskeletal filaments and use energy from ATP to move cargo along the filaments a. "Head" region (a.k.a. motor domain): Binds and binds and hydrolyzes ATP, and determines type of filament bound and direction of movement. they have an N terminus and light chains attached to the protein by a neck or hinge region. It binds to the filaments- IT DOES THE MOVING b. "Tail" region: Determines the identity of the cargo it holds, includes c-terminus •Motor proteins are around 150nm long, 2nm wide and are comprised of a coiled-coil of two alpha helices

Vesicle Targeting

• Rab Proteins: monomeric GTPases that mediate vesicle targeting • Rab-GDP is inactive, cytosolic • Rab-GTP is active, membrane bound, and recruits Rab effectors • SNAREs promote membrane fusion

Membrane Fusion

• SNARE complex brings membranes together. • Energy from formation of complex used to pull membranes together and force H 2O out. • Noncytosolic leaflets fuse and cytosolic leaflets fuse. • Bacteria that cause Tetanus and Botulism release toxins that degrade complex, so NTs not released.

Protein Translocation to ER

• SRP (Signal Recognition Particle): recognizes signal sequence to direct ribosome to the ER

Nuclear Localization Signal (NLS)

• Sequences rich in Arg and Lys, not cleaved after transport • Recognized by Nuclear Import Receptors (Importins)

Signal Sequences and Transport

• Signal Sequence: Protein sorting signal that specifies a particular destination in the cell. a. Can be 15-60 amino acids or shorter b. Commonly at the N-terminus of the protein, but can be internal c. Can also be multiple internal sequences that form a 3D signal patch • Signal Peptidases: Enzyme that removes signal when sorting is complete. -typical signal sequences in photo

Nuclear Pore Complex (NPC)

• Small molecules diffuse through NPC • Large molecules use signal sequences and receptors for transport, but usually transported in folded state.

Translocation of a Single-Pass Transmembrane Protein

• Stop Transfer Sequence anchors the protein in the membrane and changes conformation of translocator to release protein laterally into membrane.

Evolution of the nucleus and ER

• Structures pinch off from the plasma membrane • Nuclear environment similar to cytosol • ER lumen similar to extracellular space • Endosymbiosis -There is now strong evidence that nuclear pore complexes (NPCs) and nuclear membranes coevolved with the endomembrane system, and that the last eukaryotic common ancestor (LECA) had fully functional NPCs. Recent studies have identified many components of the nuclear envelope in living Opisthokonts, the eukaryotic supergroup that includes fungi and metazoan animals. These components include diverse chromatin-binding membrane proteins, and membrane proteins with adhesive lumenal domains that may have contributed to the evolution of nuclear membrane architecture. Further discoveries about the nucleoskeleton suggest that the evolution of nuclear structure was tightly coupled to genome partitioning during mitosis.

Families of Intracellular Compartments

• The nucleus and cytosol • Mitochondria • Endomembrane System (Compartments of Secretory and Endocytic pathways): ER, Golgi, Endosomes and Lysosomes • Plastids in plants (Example: Chloroplasts)

Budding and Fusion of Vesicles

• Transfer of soluble components from one lumen to another • Membrane proteins transferred, with same domain always oriented to the cytosol.

Glycosaminoglycans (GAGs)

• Unbranched polysaccharide composed of repeating disaccharide units (2 sugars). • Different GAGs composed of different sugars. • Most linked to a protein core (not hyaluronan). -help with arthritis

Skeletal Muscle Contraction: The Actin-Myosin Connection

• Voluntary movement • Separate cells (myoblasts) combine to form a muscle cell containing many nuclei. • Myofibril: subcellular unit where banding pattern is observed. A myofibril (also known as a muscle fibril) is a basic rod-like unit of a muscle cell. Muscles are composed of tubular cells called myocytes, known as muscle fibres in striated muscle, and these cells in turn contain many chains of myofibrils. -Myofibrils are made up of sarcomeres, the functional units of a muscle. The function of the myofibril is to perform muscle contraction via the sliding-filament model. When muscles are at rest, there is incomplete overlap between the thin and thick filaments, with some areas containing only one of the two types. • Sarcomere: contractile unit. Each sarcomere is composed of two main protein filaments—actin and myosin—which are the active structures responsible for muscular contraction. The most popular model that describes muscular contraction is called the sliding filament theory. -skeletal muscle is the muscle type that initiates all of our voluntary movement. Herein lies the sarcomere's main purpose. Sarcomeres are able to initiate large, sweeping movement by contracting in unison. Their unique structure allows these tiny units to coordinate our muscles' contractions. -photo includes sytructure

power stroke of dynein

• When dynein binds ATP, it releases microtubule. -head domain has individual subunits, and it has a stalk the stalk interacts with microtubules -tail domain carries cargo PHOTO SHOWS THIS

Fibronectins

• a Glycoprotein that helps cells attach to collagen in the matrix -part of it interacts with collagen, the other interacts w/ cell surface receptors called integrins • Forms dimer

basal lamina

• a.k.a. basement membrane -organizes epithelial tissues, since they are anchored within the basal lamina -detachment from basal lamina can result in skin blistering • Specialized ECM • Thin, tough sheet underlying epithelial cells • Maintains architecture of body • Mechanical connections between cells of the same tissue • In skin, detachment of epidermis from dermis can lead to diseases with increased skin blistering.

Dynein

• walks on microtubules, but it is a Minus end directed microtubule motor (plus ends grow out to membranes) -Moves things towards the middle of cell • more complex and variable structure: Composed of 2-3 heavy chains, as well as intermediate and light chains. • Two classes: CYTOPLASMIC DYENIN (vesicle trafficing/movement of organelles) and cilliary/axonemal dyenin (has protrusions capable of movemement of cilia/flagella)

+TIP Proteins

•Microtubule plus-end/positive-end tracking proteins or +TIPs are a type of microtubule associated protein (MAP) which accumulate at the plus ends of microtubules. •Modulate growth and shrinkage • Controls microtubule positioning •In addition to the basic known functions of +TIPs, the proteins are crucial for the linkages between microtubule ends and other cellular structures. +TIPs can bind microtubule ends to the cell cortex by colliding to plasma membrane-associated proteins or in the case of some +TIPs, directly to the actin fiber.

Exam 3 cell bio

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