cell molec 4

Proteoglycan

"bottle brush"-- proteoglycan "hair" -- GAGs --Glycosaminoglycans (GAGs) Proteoglycans are important for cell signaling •Heparan sulfate: present FGFs to FGFR •Perlecan : sequester BMPs

Plasmodesmata

(a) Schematic model of plasmo- desmata, showing the desmotubule, an extension of the endoplasmic reticulum (ER), and the annulus, a plasma-membrane-lined channel filled with cytosol that interconnects the cytosols of adjacent cells. The regulated deposition of a glucose polymer called callose (cyan) in the extracellular spaces in the cell wall adjacent to the entrances of the channels has the potential to block intercellular transport through the plasmodesmata, apparently by forcing the closing of the channels by narrowing the annulus. (b) Electron micrographs of thin sections of a sugarcane leaf (brackets indicate individual plasmodesmata). Left: Longitudinal view, showing ER and desmotubule running through each annulus. Right: Perpendicular cross-sectional views of plasmo- desmata, in some of which spoke structures connecting the plasma membrane to the desmotubule can be seen.

Confocal microscopy & 3D reconstruction

(achieves marginally higher resolution by imaging light from just a part of the thickness of the sample)

Match the type of cytoskeletal filament to its nucleotide binding

actin - binds ATP tubulin - binds GTP intermediate filaments - no nucleotide binding

Microfilaments are comprised of ______.

actins

Cell-cell adhesion: Tight junctions (slide 11)

(caption) (a)Freeze-fracturepreparationof tight junction zone between two intestinal epithelial cells. The fracture plane passes through the plasma membrane of one of the two adjacent cells (see also Figure 20-11). A honeycomb-like network of ridges and grooves below the microvilli constitutes the tight-junction zone. (b) Schematic drawing shows how a tight junction might be formedby the linkage of rows of protein particles in adjacent cells. In theinset micrograph of an ultrathin sectional view of a tight junction, the adjacent cells can be seen in close contact where the rows of proteins interact (a) Immunofluorescence localization of occludin (green) and tricellulin (red) in mouse intestinal epithelium. Note that tricellulin is predomi- nantly concentrated in tricellular junctions. (b) The junction adhesion molecule (JAM) has a single transmembrane domain and an extracel- lular region with two immunoglobulin domains, whereas occludin and claudins contain four transmembrane helices. The larger extracellular loop of the claudins, indicated by an asterisk, contributes to paracellular ion selectivity. The transmembrane helices of claudin-15, which permits paracellular transport of cations, form a four-helix bundle, and the extracellular loops contain a five-stranded β sheet (seen edgewise in this view). This β sheet has been proposed to help define the pore through which ions pass (near the asterisk)

Fibronectin (slide 17)

- important matrix protein in cell shape, migration and differentiation •Shape of cells •Organization of the cytoskeleton •Essential for cell migration and differentiation during embryogenesis FIGURE 2033 Organization of fibronectin and its binding to integrin. (a) Scale model of fibronectin is shown docked by two type III repeats to the extracellular domains of integrin. Only one of the two similar chains, which are linked by disulfide bonds near their C-termini, in the dimeric fibronectin molecule is shown. Each chain contains about 2446 amino acids and is composed of three types of repeating amino acid sequences (type I, II, or III repeats) or domains. The EIIIA, EIIIB— both type III repeats—and IIICS domain are variably spliced into the structure at locations indicated by arrows. Circulating fibronectin lacks EIIIA, EIIIB, or both. At least five different sequences may be present in the IIICS region as a result of alternative splicing (see Figure 5-16).Each chain contains several multi-repeat-containing regions, some of which contain specific binding sites for heparan sulfate, fibrin (a major constituent of blood clots), collagen, and cell-surface integrins. The integrin-binding domain is also known as the cell-binding domain. Structures of fibronectin's domains were determined from fragments of the molecule. (b) A high-resolution structure shows that the RGD motif (red) extends outward in a loop from its compact type III domain on the same side of fibronectin as the synergy region (blue), which also contributes to high-affinity binding to integrins.

Defective cell-cell interaction in disease

-- another type of cell adhesion mediated by ICAM (Ig superfamily) and P-selectin. (slide 27 caption) Endothelium-leukocyte interactions: activation, binding, rolling, and extravasation. Step 1 : In the absence of inflammation or infection, leukocytes and endothelial cells lining blood vessels are in a resting state and not interacting. Step 2 : Inflam- matory signals released only in areas of inflammation, infection, or both activate resting endothelial cells, resulting in the movement of vesicle-sequestered selectins to the cell surface. The exposed selectins mediate weak binding of leukocytes by interacting with carbohydrate ligands on leukocytes. Blood flow forces the loosely bound leukocytes to roll along the endothelial surface of the blood vessel (curved arrow).Activation of the endothelium also causes synthesis of platelet- activating factor (PAF) and ICAM-1, both expressed on the endothelial cell surface. PAF and other, usually secreted, activators, including chemokines, then induce changes in the shapes of the leukocytes and activation of leukocyte integrins such as αLβ2, which is expressedby T lymphocytes (step 3 ). The subsequent tight binding between activated integrins on leukocytes and CAMs on the endothelium (e.g., ICAM-2 and ICAM-1) results in firm adhesion (step 4 ) and subsequent movement (extravasation) into the underlying tissue (step 5 ).

what are the advantages of cryo-EM compared to traditional transmission EM? Select all that apply.

-Cryo-EM images samples in their native, hydrated state -Cryo-EM does not require staining or coating with heavy metals

Match each protein with its appropriate description

-Type IV collagen - a trimeric protein with a triple-helix structure, that forms a two-dimensional sheet -laminin - a heterotrimeric protein shaped like a cross and binds to integrins -perlecan - large multidomain proteoglylcan that cross-links ECM components and cell surface molecules -nidogen - a rod-like molecule that cross-links type IV -collagen, perlecan and laminin

Plasmodesmata directly connect _______ of adjacent plant cells.

-cytosols -endoplasmic reticulum extensions between the two cells

Which of the following would yield better resolution (smaller value of D)? Select all that apply.

-immersion of the objective in oil (refractive index = 1.56) instead of exposed to air (refractive index = 1) -an objective with a shorter focal length (designed to work closer to the specimen)

what is needed in the culture medium of animal cells? select all that are minimally necessary for all animal cell cultures

-nutrients such as glucose -growth factors -any amino acids or vitamins that the cells cannot make themselves

Which of the following statements about αβ-tubulin dimers and their assembly into microtubules is correct?

-they assemble into microtubules in a polarized manner. -GTP is bound to α-tubulin and is non-exchangeable. -GTP is bound to β-tubulin, but after assembling into microtubules, it is hydrolyzed to GDP. -The nucleotide bound by β-tubulin is exchangeable.

What salts should be in a mammalian cell culture medium?

0.15 M NaCl (as in blood) 0.15 M NaCl is isotonic for mammalian cells, and is the major salt in physiological saline solutions used for IV drips.

Transmission and Scanning Electron Microscopy

1.Small molecule/structure: negative stain or metal shadowing -> TEM 2.Larger tissue: section+ metal -> TEMmetal-shadowing -> SEM 3.Live sample: frozen -> cryoEM

Gap junctions

Adhesion Type- cell- cell Principal CAMs or Adhesion Receptors- Connexins, innexins, pannexins Cytoskeletal Attachment- Via adapters to other junctions Intracellular Adapters- ZO-1,2,3 Function- Communication, small-molecule trans- port between cells

Tight junctions

Adhesion Type- cell- cell Principal CAMs or Adhesion Receptors- Occludin, claudins, JAMs Cytoskeletal Attachment- Actin filaments Intracellular Adapters- ZO-1,2,3, PAR3, cingulin Function- Controlling solute flow, signaling

Plasmodesmata (plants only)

Adhesion Type- cell- cell Principal CAMs or Adhesion Receptors- Undefined Cytoskeletal Attachment- Actin filaments Intracellular Adapters- NET1A Function- Communication, molecule transport between cells

Which of the protein below is present in lamellipodia but not in stress fibers?

Arp2/3

Integrin mediates diverse cell functions (slide 15 caption)

Binding of integrins to their ligands induces conformational changes in their cytoplas- mic domains, directly or indirectly altering their interactions with cytoplasmic proteins (outside-in signaling). These cytoplasmic proteins include adapter proteins (e.g., talins, kindlins, paxillin, vincu- lin) and signaling kinases [Src-family kinases, focal adhesion kinase (FAK), integrin-linked kinase (ILK)] that transmit signals via diverse signaling pathways, thereby influencing cell proliferation, cell survival, cytoskeletal organization, cell migration, and gene transcription. Components of several signaling pathways, some of which are associated directly with the plasma membrane, are shown in green boxes. Many of the components of the pathways shown here are shared with other cell-surface- activated signaling pathways (e.g., receptor tyrosine kinases shown on the right) and are discussed in Chapters 15 and 16. In turn, intracellular signaling pathways can, via adapter proteins, modify the ability of integrins to bind to their extracellular ligands (inside-out signaling).

Experiment showing that fibronectin is important for tissue morphogenesis

Branching morphogenesis of salivary glands Antibodiestofibronectinblock branching morphogenesis in developing mouse tissues. Immature salivary glands were isolated from murine embryos and allowed to undergo branching morphogenesis in vitro for 10 hours in the absence (a) or presence (b) of an antibody that binds to and blocks the activity of the ECM molecule fibronectin. Anti-fibronectin antibody (Anti-FN) treat- ment blocked branch formation (arrowheads). Inhibition of fibronec- tin's adhesion receptor (an integrin) also blocks branch formation (not shown). Scale bar, 100 μm.

Cell-cell adhesion: Gap junctions (slide 12)

CHANNELS mediating cell-cell communication:Connexin (6 subunits)Allow transport of small molecules, e.g. Ions, cAMP, etc.Phosphorylation of connexins can modulate the permeability of gap junctions (caption) Gap junctions. (a) In this thin section through a gap junction connecting two mouse liver cells, the two plasma membranes are closely associated for a distance of several hundred nanometers, separated by a "gap" of 2-3 nm. (b) Numerous roughly hexagonal particles are visible in this perpendicular view of the cytosolic face of a region of plasma membrane enriched in gap junctions. Each hexagonal particle aligns with a similar particle on an adjacent cell, forming a channel connecting the two cells. (c) Schematic model of a gap junction connecting two plasma membranes. Both membranes contain connexon hemichannels, cylinders of six dumbbell-shaped connexin molecules. Two connexons join in the gap between the cells to form a gap-junction channel, 1.4-2.0 nm in diameter, that connects the cytosols of the two cells. (d) Structure of recombinant human Cx26 gap junction as determined by x-ray crystallography (3.5-Å resolution). Left: Space-filling model of a side view of the complete structure of two attached connexons oriented as in part (c). Each of the six connexins that comprise a connexon has four transmembrane helices and is shown in a distinct color. The structures of the loops connecting the transmembrane helices are not well defined and not shown.

Transformed or stem cell lines

Can grow in liquid suspension "Immortal" - can be cultured indefinitely Not contact-inhibited; form 3-D mounds or balls Relatively undifferentiated

Actin treadmilling

Cc= Concentration of free G-actin at which the assembly onto a filament end is balanced by loss from that end

Double-label fluorescence microscopy can visualize the relative distributions of two proteins

Cells (blue) taking up exosomes (green)

Which of the following is found in a proteoglycan?

Chondroitin sulfate

Actin-binding proteins regulate the assembly and turnover of actin filament

Cofilin ->depolymerization Profilin-> polymerization Thymosin:reservoir for G-actin ->Inhibits polymerization

Which protein forms the gap juctions?

Connexin

Light microscopy

DIC and phase contrast microscopy utilize differences in refractive index to enhance contrast

Visualization Methods -- Optical/Light Microscopy

DIC, phase-contrast Fluorescent Microscopy, confocal microscopy Super-resolution Microscopy Light-Sheet Microscopy

To separate organelles that are close in size, one employs the technique called sucrose gradient centrifugation. This technique separates organelles by:

Density

Cell-cell adhesion: Desmosomes

Difference between desmosome and adherens junction? Desmosomes (Figure 20-16) con- tain two specialized cadherins, desmoglein and desmocollin, whose cytosolic domains are distinct from those in the classical cadherins. The cytosolic domains of desmosomal cadherins bind to adapter proteins such as plakoglobin (simi- lar in structure to β-catenin) and plakophilins, and these bind to a member of the plakin family of adapters, called desmo- plakin. These adapters form the thick cytoplasmic plaques that are characteristic of desmosomes. The desmoplakins directly mediate plaque binding to intermediate filaments.

Which motor protein mediates the beating of cilia and flagella?

Dynein

Connective tissue

Elastic and collagen fibers in connective tissue. (a) Light-microscopic image of loose connective tissue from the lung. Elastic fibers are the thin fibers that are stained purple, collagen fibers (bundles of collagen fibrils) are stained pink, and the nuclei of cells are stained purple. (b) Longitudinal and (c) cross-sectional electron micro- scopic images of elastic fibers and collagen fibrils (coll) in the skin of a mouse. The elastic fibers have a solid core of elastin (e) integrated into and surrounded by a bundle of microfibrils (mf). Scale bars, 0.25 μm

Cryo-electron tomography

Enhanced 3-D image from averaging thousands of images of nucleopores

what is the name of the protein kinase that is activated at focal adhesions? Use the 3 letter initials for the name rather than writing it out.

FAK

Sheet-forming Collagen: collagen layer in basal lamina

FIGURE 2026 Structure and assembly of type IV collagen.(a) Schematic representation of type IV collagen. This 400-nm-long molecule has a small noncollagenous globular domain at the N-terminus and a large globular domain at the C-terminus. The collag- enous triple helix is interrupted by nonhelical segments that introduce flexible kinks into the molecule. Lateral interactions between triple- helical segments, as well as head-to-head and tail-to-tail interactions between the globular domains, form dimers, tetramers, and higher- order complexes, yielding a sheet-like network. Multiple, unusual sulfilimine (-S=N-) or thioether bonds between hydroxylysine (or lysine) and methionine residues covalently cross-link some adjacent C-terminal domains and contribute to the stability of the network. See A. Boutaud, 2000, J. Biol. Chem. 275:30716. (b) Electron micrograph of type IV collagen network formed in vitro. The lacy appearance results from the flexibility of the molecule, the side-to-side binding between triple-helical segments (white arrows), and the interactions between C-terminal globular domains (yellow arrows).

Actin assembly is nucleated by two classes of proteins, ___ for unbranched filaments and ____for branched filaments.

Formin and Arp 2/3 complex

Laminin: heterotrimeric multi-adhesive matrix protein

Globular domains bind to various adhesion receptors and matrix molecules (a) Schematic model of cross- shaped laminin molecule showing the general shape, location of glob- ular domains, and coiled-coil region in which laminin's three chainsare covalently linked by several disulfide bonds. Different regions of laminin bind to adhesion receptors and various matrix components (indicated by arrows). Right: Laminins assemble into a lattice via interac- tions between their N-terminal globular domains. See G. R. Martin and Laminins (self-assembly), collagen FIGURE 2024 10 nm R. Timpl, 1987, Annu. Rev. Cell Biol. 3:57; M. Durbeej, 2010, Cell Tissue Res. 339:259-268; and S. Meinen et al., 2007, J. Cell Biol. 176:979-993. (b) Electron micrographs of an intact laminin molecule, showing its characteristic cross shape (left), and the carbohydrate-binding LG do- mains near the C-terminus (right)

Actin Filaments

Helical twist: 28 subunits in each repeating unit

If you wanted to isolate just vesicles coated with COPI protein from cells, what method would give you the cleanest results?

Immuno-affinity purification with antibodies to COPI coat protein

ECM proteins are required for normal development

Inactivating the genes for some ECM proteins results in defective skeletal development in mice. These photographs show skeletons of normal (left), collagen II- deficient (center), and perlecan-deficient (right) murine embryos that were isolated and stained to visualize the cartilage (blue) and bone (red). Absence of these key ECM components leads to dwarfism, with many skeletal elements shortened and disfigured.

Integrin

Integrins function as adhesion receptors and CAMs in a wide variety of epithelial and nonepithelial cells, mediating many cell-matrix and cell-cell interactions (Table 20-4). In vertebrates, at least 24 integrin heterodimers, composed of 18 types of α subunits and 8 types of β subunits in various αβ heterodimeric combinations, are known. A single type of β chain can interact with any one of several different types of α chains, forming distinct integrins that bind different ligands. This phenomenon of combinatorial diversity allows a relatively small number of components to serve a large number of distinct functions. Although most cells express several distinct integrins that bind the same or different ligands, many integrins are expressed predominantly in certain types of cells. Not only do many integrins bind more than one ligand, but there are ligands that can bind to any one of several different integrins. All integrins appear to have evolved from two ancient general subgroups: those that bind proteins containing the tripeptide sequence Arg-Gly-Asp, usually called the RGD motif (fibronectin is one such protein), and those that bind laminin. Several integrin α subunits contain a distinctive in- serted domain, the I-domain, which can mediate binding of certain integrins to various collagens in the ECM. Some inte- grins with I-domains are expressed exclusively on leukocytes (white blood cells) and red and white blood cell precursor (hematopoietic) cells. I-domains also recognize CAMs on other cells, including members of the Ig superfamily (e.g., ICAMs, VCAMs), and thus participate in cell-cell adhesion.

Further purification & identification of organelles, vesicles using antibodies (immunoaffinity purification)

Staphylococcus aureus has protein A on cell surface

Which of the following proteins facilitate microtubule disassembly?

Kinesin-13

Which of the following is NOT a component of the extracellular matrix?

Lamin

Basal lamina

Major protein components of the basal lamina. Type IV collagen and laminin each form two-dimensional networks (see Figures 20-24 and 20-26), which are cross-linked by nidogen/entactin and perlecan molecules and which interact via laminins with the plasma membranes of adjacent cells. A basal lamina separates epithelial cells and some other cells from connective tissue. (a) Transmission electron micrograph of a thin section of cells (top) and underlying connec- tive tissue (bottom). The electron-dense layer of the basal lamina can be seen to follow the undulations of the basal surfaces of the cells. (b) Electron micrograph of a quick-freeze deep-etch preparation of skeletal muscle, showing the plasma membrane, basal lamina, and surrounding connective-tissue collagen fibers. In this preparation, the basal lamina is revealed as a meshwork of filamentous proteins that associates with the plasma membrane and the thicker collagen fibers of the connective tissue.

Cell-Cell Adhesion

Microvillus Tight junction Adherens junction Actin and myosin filaments Gap junction Intermediate filaments Desmosome Hemidesmosome Basal lamina Connective tissue (page 974 in textbook)

Fluorescence Microscopy

Most versatile and powerful technique for localizing molecules within a cell by light microscopy.Use molecular genetics approaches to express a fusion between a protein of interest and a naturally fluorescent protein.Localize and quantify specific molecules in living cells.Use fluorophores- molecules that absorb and emit light at specific wavelengths. Many different colors of fluorescent proteins are now available

Which of the folllowing motor proteins travel on actin filaments?

Myosin II Myosin IV

Actin assembly has three steps, nucleation, elongation, and steady state. Which step takes the most of the time to occur?

Nucleation

Experiments showing E-cadherin mediates cell-cell adhesion, which depends on Ca2+

Under standard cell culture conditions, in the presence of calcium in the extracellular fluid, L cells do not aggre- gate into sheets (left). Introduction of a gene that causes the expression of E-cadherin in these cells results in their aggregation into epithelium- like clumps in the presence of calcium (center), but not in its absence

Stochastic Optical Reconstruction Microscopy (STORM)

Very low excitation light intensity

Which of the following accelerates polymerization of a microfilament on the + end?

Profilin Formin

Extracellular matrix proteins

Proteoglycans (cushioning) - Perlecan Collagens (integrity and strength) - Sheet forming (e.g., type IV) , Fibrillar collagens (e.g., types I, II, and III) Multi-adhesive matrix proteins (cell-matrix adhesion, cell shape and behavior) - Laminin, Fibronectin, Nidogen/entactin Cell anchorage; tissue property; cell polarity, fate, survival, proliferation, and differentiation; cell migration; concentration of signal molecules; ligand-receptor interaction.

Integrins bind to what sequence in fibronectin?

RGD

Light Microscopy

Resolution/Diffraction limit (D) •The ability to distinguish between two closely positioned objects D = 0.61λ/Nsinα (Nsinα = NA) •N= refractive index of medium between specimen and objective lens (1 for air, 1.33 for water, 1.56 for oil) •λ =Wavelength of light (major factor that limits D in light microscopy) •α = half angle of the cone of light that enters the objective Objectives: 10x/0.25, 20x/0.40, 40x/0.64 oil, 60x/1.30 oil, etc.

List the fundamental requirements for culturing animal (mammalian) cells

Salts & buffering agentsNutrients•Glucose•Vitamins•Essential amino acidsGrowth factors•Serum from fetal calves•Serum-free media has insulin, transferrin + growth factors

Plant cell

Structure of the plant cell wall. (a) Overview of the organization of a typical plant cell, in which the organelle-filled cell with its plasma membrane is surrounded by a well-defined extra- cellular matrix called the cell wall. (b) Schematic representation of the cell wall of an onion. Cellulose and hemicellulose are arranged into at least three layers in a matrix of pectin. The sizes of the polymers and their separations are drawn to scale. To simplify the diagram, most of the hemicellulose cross-links and other matrix constituents (e.g., extensin, lignin) are not shown (c) Quick-freeze deep-etch electron micrograph of the cell wall of a garden pea in which some of the pectin molecules were removed by chemical treatment. The abun- dant thicker fibers are cellulose microfibrils, and the thinner fibers are hemicellulose cross-links (red arrowheads).

α4β1 is the major integrin isoform in T cells, which can binds to VCAM.Binding of wt α4 or mutant α4 (Y991A) to VCAM-coated beads were quantified in figure (a). Bio1211 is a specific inhibitor for binding of α4β1 to VCAM. Amino acid 991 locates in the cytoplasmic domain of α4Figure (b) shows light microscopic images of T cells allowed to settle onto a substrate composed of VCAM for 1 minute and then subjected to a shear flow for 10 minutes. What can you deduce of the mutation from these observations?

The mutation Y991A in the α4 subunit prevents α4 from binding to paxillin, a component of the actin cytoskeleton. Control siRNA or paxillin siRNA is applied to cells expressing wild-type or Y991A α4 and the cells are allowed to settle onto a substrate of VCAM and then subjected to a shear force. The percentage of cells bind to substrate under different shear stress are shown in the graph below. What information do these data provide about how adhesion of T cells to their substrate is mediated?

Actin Polymerization

The plus end: fast growing The minus end: slow growingActin filament decorated by myosin to determine orientation.

Isolation and Characterization of Cell Organelles

To purify the contents of the cell, first break through the wall and plasma membrane. •French press with hypotonic solution •Sonication•Enzymes (lysozyme) and detergent •Bead beating (microscopic glass, ceramic, steel beads)

Which type of collagen is responsible for forming a sheet, not fibrils?

Type IV

Match each named junction to the prinicpal CAM or adhesion receptor

adherens junctions and desmosomes - Cadherins Hemidesmosomes - Integrins tight junctions - occulins, claudins, JAMs gap junctions - connexins

Anchoring junctions 1. Adherens junctions 2.Desmosomes 3. Hemidesmosomes 4. Focal, fibrillar, and 3-D adhe- sions

adhesive type- cell-cell Principal CAMs or Adhesion Receptors- 1. Cadherins 2. Desmosomal cadherins 3. Integrin (α6β4) 4. Integrins Cytoskeletal Attachment 1. Actin filaments 2. Intermediate filaments 3. Intermediate filaments 4. Actin filaments Intracellular Adapters 1. Catenins, vinculin 2. Plakoglobin, plako- philins, desmoplakins 3. Plectin, dystonin/ BPAG1 4. Talin, kindlin, paxil- lin, vinculin kinase Function 1. Shape, tension, signal- ing, force transmission 2. Strength, durability, signaling 3. Shape, rigidity, signaling 4. Shape, signaling, force transmission, cell movement

organization of tissue

apical (top) epithelium (tight/adherens/gap junctions) MET basal (bottom) endothelium (epithelium) (adherens/gap junction) EMT mesenchyme cell-matrix adhesion apical-- smooth muscle intestine epithelium basal

Which of the following are functions of fibronectin?

binding of cell to extracellular matrix cellular migration cellular differentiation cytoskeletal organization and cell shape mechanotransduction

Cell-cell adhesion: Adherens junctions (page 977)

caderins (when these from two different cells connect its trans but when it connected to itself it cis) they connect in the extracellular space (in the cytoplasm lots of stuff is going on check text)

Match the cell adhesion molecule to its correct description

cadherins - Ca2+-binding cell-cell adhesion molecule integrins - heterodimeric cell surface molecules responsible for adhesion to substrate Ig domain molecules - usually monomeric, mediate diverse cell-cell recognition and cell attachment interactions selectins - recognize and bind carbohydrates

List requirements for a cell to become part of a tissue

cell-cell adhesion (cells stick together) cell-substrate adhesion (cells attach to something) polarity (which way is up?) cell-cell communication (what am I supposed to do, and what are my neighbors doing?) cell-cell or cell-matrix recognition (where am I?)

Which of the following does has MTOC activity?

centrosome spindle pole basal body

Which glycosaminoglycan (GAG) is part of a proteoglycan?

chondroitin sulfate heparin sulfate karatan sulfate

Match each protein to its function

cofilin - depolymerizes microfilaments at minus end profilin - promotes actin polymerization thymosin - inhibits actin polymerization (maintains reservoir of G-actin)

Cell adhesion molecules (CAMs)

homophillic interactions- caderins (e- on one cell extend with caderin domains and have calcium binding sites connecting the second cell's cadherin domains cadherin) lg- superfamily CAMS (NCAM)- lg domains attach between cells (looks like 2 are connected) heterophilic interactions- integrins (avb3) connect to fibronectin selectins(P-selectin) the lectin domain connects to the sugars from the glycoprotiens on the other cell

integrin activation

inactive, low affinity integrin is bent active, high affinity integrin is extended and has talin binding site and kindlin binding site on beta strand

Cell-matrix adhesion:

integrin links ECM and cytoskeleton through Focal Adhesion

In fluorescence microscopy, the sample is excited by absorption of light at certain wavelengths. The sample then emits light at

longer wavelength

Which of the following cytoskeletal filaments are associated with the motor proteins?

microfilaments microtubules

Match the cytoskeletal element to its description

microfilaments - cortical networks, contraction microtubules - long-range organelle transport, cellular organization intermediate filaments - integrity of cell structures and tissues

Connects the types of cytoskeleton with their function.

microfilaments - membraen protrusion and cell migration microtubules - chromosome separation during mitosis intermediate filaments - the integrity of nucleus

The Components of the Cytoskeleton

microfilaments-- Actin (7-9 nm) microtubules-- ab- tubulin dimer(25 nm) intermediate filaments-- varies (10nm)

EMT is mediated by changes in cadherins

neural crest cells leaving neural epithelium (neural tube) metastatic cancer cells leave their primary site and invade new tissue (EMT: Epithelial to Mesenchymal Transition) A protein called Snail that suppresses the expression of E-cadherin is associated with the epithelial-to-mesenchymal transition (EMT). (a) Normal epithelial MDCK cells grown in culture. (b) Expression of the snail gene in MDCK cells causes them to undergo an EMT.(c) Distribution of E-cadherin detected by immunohistochemical staining (dark brown) in thin sections of tissue from a patient with hereditary diffuse gastric cancer. E-cadherin is seen at the intercellular borders of normal stomach gastric gland epithelial cells (right); no E-cadherin is seen at the borders of underlying invasive carcinoma cells. (979 for reference)

Immunogold labeling can be used to detect specific structure/protein

peroxisomes-- little dots

Which end of an actin filament as the lower critical concentration?

plus end

Because Myosin II is moving towards the ____ end of actin filaments, the orientation of actomyosin contractile bundles is orientated with (a) as ____ end and (b) as ____end.

plus, minus, minus In striated muscle, the actin filaments are anchored to the Z-disks at their plus ends. The Z-disks thus cap the plus ends and stabilize the actin filaments. The concentration of free actin (G-actin) is below the critical concentration of the minus end (Cc-). The myosin motors pulling towards the plus ends then results in contraction, bringing the Z-disks closer together.

match types of microtubules in mitotic spindle to their associated motor proteins.

polar MT - kinesin 5 astral MT - dynein kinetochore MT - kinesin 13

Match the kinesin structure to its function.

polarized kinesin with a moter-containing head and a tail with protein-binding domain - organelle transport bipolar kinesin with motor domains on both ends - microtubule sliding Moter-containing head with a short stalk, no tail - microtubule end disassembly

Which of the following contribute to the treadmilling of actin filaments?

profilin cofilin

primary culture

require attachment Senescence after limited number of cell divisions Contact inhibited; grow as monolayers on culture dishes Differentiated cell types; may require 3-D structure support (extracellular matrix) or "feeder" layers of cells

Which type of electron microscopy is used to detect surface feature of samples?

scanning electron microscopy (SEM)

Where is hyaluronan found in the body? Select all that apply

surrounding migrating and proliferating cells cartilage joints

Which layer of the basal lamina is closes to the plasma membrane of an epithelial cell?

the sheet of laminin and other associated proteins

What limits the resolution, D, of ordinary light microscopy?

the wavelength of visible light

Match the junction to its function

tight junction - seals off body cavities, controls paracellular flow of ions and small molecules gap junction - connects cytoplasm of adjacent cells, regulates flow of solutes and ions between cells adherens junction - located near apical surface, associated with circumferential belt of actin and myosin filaments to control cell shape desmosomes - located on lateral surface, associated with intermediate filaments, form spot contacts between cells (like rivets) hemidesmosomes - located on basal surfaces, associated with intermediate filaments, anchors cell to ECM (like nails holding down a carpet)

When cells in culture are prepared for passaging Ii.e., released from binding to the dish and each other) they are usually washed with EDTA, a reagent that binds calcium. why do you think this is done?

to release cadherins, which require calcium to bind to each other

Which imaging technique has the best resolution (smallest D)?

transmission electron microscopy

Visualization Methods -- Electron Microscopy

transmission electron microscopy scanning electron microscopy cryo-electron microscopy

Fibrillar Collagens

type 1 collagen fibrils (in tendons) type 2 collagen fibrils (in cartilage) (a) In tendons, type I fibrils are all oriented in the direction of the stress applied to the tendon. Proteoglycans and typeVI collagen bind noncovalently to type I fibrils, coating the surface. The microfibrils of type VI collagen, which contain globular and triple-heli- cal segments, bind to type I fibrils and link them together into thicker

When do leukocytes bind tightly to endothelial cells during extravasation?

when leukocyte integrins are activated and bind ICAMs

Cryo-Electron Microscopy

•Biological samples preserved in native state, frozen in vitrified water, stable even in high vacuum •Low energy electrons preserve sample, no need for staining or coating with heavy metals •Direct electron detectors for high sensitivity •For uniform particles, can average tens of thousands of images for detailed imaging & 3-D reconstruction

Immunofluorescence Microscopy

•Can detect specific proteins in fixed cells •Uses antibody that has a fluorescent dye attached to it covalently .•Generate antibody to protein of interest •Purify the antibody •Use the antibody to locate protein of interest within fixed cells/tissue •Antibody can be labeled with fluorescent compound •Or use secondary antibody (antibody that binds another antibody) labeled with fluorescent compound

Centrifugation of cell lysates

•Differential centrifugation - common first step •Sucrose gradient centrifugation- separates organelles by density

Overview of Actin Structures and their Functions

•Globular actins (G-actin) assemble into filamentous actin (F-actin) •F-actin further organizes into structures: tight bundles (short: filopodia, microvilli; long: stress fiber, contractile ring); meshwork (lamellipodia). •Various actin binding proteins coordinate in the formation of different structures.

Limitations of traditional EM

•Sample is dried •High vacuum required •High energy electron beam will destroy biological samples, so have to stain with heavy metals: metal shadowing.