Introduction to Cell and Ectracellular Matrix Ultrastructure

Question 1

What three components make up the cytoskeleton of the cell? What are the roles of each? Which ones show polarity, which does not?

Answer 1

1. Microfilaments (actin)
   
   1. 7nm thick
   2. Microfilaments are assemblies of actin monomers
   3. Microfilaments span cells and attach to cell membrane via junctional complexes
   4. Support cell shape (stress fibrils)
   5. Participate in cell locomotion
   6. Anchor organelles and proteins
   7. Transport organelles and proteins
   8. Participate in cell divisions; contractile ring
2. Intermediate filaments (vimentin, cytokeratin)
   
   1. 8-10 nm thick
   2. Formed by several proteins:
      
      1. In epithelium –> Keratins
      2. In mesenchyme –> vimentin (fibroblasts, osteoblasts), desmin (muscles), periferin (nerves)
   3. Structural support (tonofilaments)
   4. Participate in the cell-cell contacts creating intercellular tonofilament networks.
3. Microtubules (tubulin)
   
   1. 25nm thick
   2. Microtubules are assemblies of tubulin dimers
   3. Individual microtubules form networks, or form complexes such as centrioles and cilia/flagella
   4. Support cell shape (part of the cytoskeleton)
   5. Major cellular molecular motors; essential for cell division and motility
   6. Participate in organelle transport
   7. Show polarity; contains + end and - end

Question 2

How are intermediate filaments related to cell or tissue type?

Answer 2

1. In epithelium: keratins
2. In mesenchyme:
   
   1. Vimentin (fibroblasts, osteoblasts),
   2. Desmin (muscles),
   3. Periferin (nerves)

Question 3

Describe different cell compartments. What are the roles of Golgi apparatus, rough and smooth endoplasmic reticulum and mitochondria?

Answer 3

1. Golgi apparatus
   
   1. Post-translational modifications, folding, and sorting
2. Smooth and Rough Endoplasmic Reticulum
   
   1. Smooth ER: lipid synthesis
   2. Rough ER: protein synthesis
3. Mitochondria
   
   1. Energy supply

Question 4

What can the relative abundance of different intracellular compartments tell us about the physiological status of the cell?

Answer 4

1. Abundance of golgi means that the cells are most likely secretory
2. Abudance of smooth ER means there is extensive lipid metabolism occuring inside the cell
3. Abundance of rough ER (along with golci) means that the cell is producing a lot of proteins that are either to be utilized by the cell itself or to be secreted out of the cell e.g. osteoblast (osteocyte however does not have as much rough ER)
4. Abundance of mitochondria means that the cells require extra amount of energy to carry out its role
   
   1. e.g. Miocyte in muslce cells

Question 5

Categorize the various types of cell junctions by functions and attachment.

Answer 5

1. Tight Junctions (Zonula occludens)
   
   1. Form chemically impermeable contacts, preventing diffusion of ions through the extracellular space
2. Adherens Junctions (Zonula adherens)
   
   1. Form reversible cell-cell and cell-extracellular matrix (ECM) contacts.
   2. Ca-dependent, wider than tight junctions
   3. Linked to actin skeleton
3. Desmosomes (Macula adherens)
   
   1. Form mechanically strong contacts between cells, often anchor intermediate filaments e.g. tonofilaments
   2. Desmosomes in stratum spinosum of oral mucosa are believed to hold together epithelium and prevent diffusion of exogenous compounds and pathogen
   3. Composed of Desmoplakin, pakoglobin, desmoglein, desmocollin, membrane, and intermediate filaments
4. Hemidesmosomes
   
   1. Form mechanically strong contacts between cells and extracellular structures, i.e. basal lamina
   2. Composed of keratin filaments (intermediate filaments), intracellular plaque, membrane, basal lamina (made of lamina lucida & lamina densa), and anchoring fibrils

Question 6

What is the difference between a zonula and macula?

Answer 6

1. Zonula = belt type contact circumferential to the cell
2. Macula = spot type contact

Question 7

What are the components of intercellular junctions and what are their roles in the process of attachment?

Answer 7

1. Gap Junctions
   
   1. Intercellular pores composed of connexons (made of connexins building block)
   2. Found between 2 osteoblasts
   3. Transmit signals between bone cells including network of osteocyte
2. Desmosomes provide strong mechanical bonding between the cells, preventing extracellular diffusion
3. Hemidesmosomes form mechanically robust cell-ECM contacts
4. Cell-cell and Cell-ECM junctions are involved in cell signaling

Question 8

How are tight junctions related to cell polarity? What types of transmembrane proteins are associated with them?

Answer 8

1. The “tightness” of the junction to water and ions (especially cations) is related to the specific claudin(s) present and is correlated with the number of strands of transmembrane proteins.
2. Several cytoplasmic proteins associate with the intracellular portions of the transmembrane proteins;
   
   1. cell polarity–related proteins
   2. vesicular transport–related proteins
   3. kinases
   4. transcription factors
   5. a tumor suppressor protein.

Question 9

How are junctions important in cell signaling?

Answer 9

1. Direct contact between cells allows the receptors on one cell to bind the small molecules attached to the plasma membrane of different cell.
2. Gap junctions, specifically, are the main site of cell-cell signaling or communication that allow small molecules to diffuse between adjacent cells

Question 10

What are integrins? Are they involved in signaling?

Answer 10

1. In focal adhesions the transmembrane component is a member of the integrin family of adhesion molecules.
2. Ligand binding by integrins also leads to the recruitment and activation of various intracellular signaling molecules, including guanine nucleotide–binding proteins and several protein kinases

Question 11

Describe the structure and components of a basement membrane and its attachment to the cell? Are extracellular proteins important in this attachment? If so, which ones?

Answer 11

1. The basement membrane is the fusion of two laminae, the basal lamina and the reticular lamina (or lamina reticularis).
2. Consists of two structural components, the lamina lucida, adjacent to the basal cell membrane, and the lamina densa, between the lamina lucida and the connective tissue
3. The main constituents of the basal lamina are type IV collagen, which forms a “chicken-wire” network:
   
   1. Adhesive glycoprotein laminin
   2. Heparan sulfate proteoglycan

Question 12

What are some of the functions of the basal lamina?

Answer 12

1. The basal lamina, along with hemidesmosomes, attaches the epithelium to the underlying connective tissue, functions as a filter to control the passage of molecules between the epithelium and connective tissue, and acts as a barrier to cell migration. The basal lamina also has important signaling functions, which are essential for epithelial differentiation and the development and maintenance of cell polarity.
2. Consists of two structural components, the lamina lucida, adjacent to the basal cell membrane, and the lamina densa, between the lamina lucida and the connective tissue
3. In epithelia, there is a third layer, the lamina fibroreticularis, closely associated with the lamina densa.
4. The main constituents of the basal lamina are type IV collagen, which forms a “chicken-wire” network;
   
   1. the adhesive glycoprotein laminin;
   2. a heparan sulfate proteoglycan.
5. Fibronectin, an adhesive glycoprotein, type III collagen (reticular fibers), type VII collagen (anchoring fibrils), and other types of collagen all made by fibroblasts are present in the lamina fibroreticularis and help maintain the attachment of the basal lamina to the underlying connective tissue.

Question 13

Are all fibroblasts the same? Do they exhibit cell-cell attachments?

Answer 13

1. The degree of synthetic and secretory capacity of fibroblasts is evidenced by the amount of rough endoplasmic reticulum, secretory granules, and mitochondria, and the extent of the Golgi complex in their cytoplasm
2. Although fibroblasts of different tissues have similar appearances, distinguishable mainly as active or quiescent, considerable heterogeneity exists within fibroblast populations.
3. This heterogeneity is manifested as differences in their synthetic products, rates of synthesis and turnover, response to regulatory molecules, proliferation rates, and others.
4. In most connective tissues, fibroblasts are separated from one another by the extracellular matrix components; therefore, intercellular junctions are NOT present.
5. Exceptions are embryonic tissue, in which gap junctions occur frequently, and the periodontal ligament, in which fibroblasts frequently exhibit cell-to-cell contacts of the adherens type.
6. Fibroblasts also form specialized focal contacts with components of the extracellular matrix

Question 14

Describe the steps in the synthesis of a type I collagen fiber. Which processes are intra cellular, which are extracellular?

Answer 14

1. As a secretory protein, fibrous collagen is synthesized as a proprotein (procollagen) in a manner similar to secretory proteins of other cells; they have N- and C-terminal extensions that are important for assembly of the triple-helical molecule they are translocated into the cisternae of the rough endoplasmic reticulum, where some posttranslational modifications occur
2. The first modification is hydroxylation of many of the proline and lysine residues in the chain, which permits hydrogen bonding with the adjacent chains as the triple helix is assembled. The vitamin C–dependent enzymes prolylhydroxylase and lysylhydroxylase are required for this step.
3. Proper alignment of the chains in a triple helix is achieved
   by disulfide bonding at the C-terminal extension
4. The three chains then twist around themselves to “weave” the helix. The assembled helix is transported through the Golgi complex where glycosylation is completed by the addition of glucose to the O-linked galactose residues
5. Such coiling as occurring in the rough endoplasmic reticulum where folding and cross-linking enzymes reside
6. Secretory granules containing the procollagen molecules are formed at the trans face of the Golgi complex and are released subsequently by exocytosis at the cell surface.
7. The formation of typical banded collagen fibrils occurs
   extracellularly. The C-terminal extensions, and at least part of the N-terminal ones, are removed by the action of C- and N-proteinases as the molecules are about to be secreted and/or also extracellularly soon after
   their release
8. After the fibrils are assembled, the remaining portions of the N-terminal extensions are removed by procollagen peptidase.
9. The oxidation of some lysine and hydroxylysine residues by the extracellular enzyme lysyl oxidase results in intermolecular cross-links that further stabilize the fibrils.

Question 15

How are the various types of collagen classified? Are some directly (integrally) linked to the cell surface?

Answer 15

1. The collagen superfamily contains at least 27 types of collagens that together constitute the most abundant proteins found in the body
2. All collagens are composed of three polypeptide alpha chains coiled around each other to form the typical collagen triple-helix configuration
3. Variations among the collagens include differences in the assembly of the basic polypeptide chains, lengths of the triple helix, interruptions in the helix, and the terminations of the helical domains.
4. Fibrillar collagens, basal lamina collagen, fibril-associated collagens with interrupted triple helices, network-forming collagens, anchoring-fibril collagen, microfibril-forming collagen, transmembrane collagen, multiplexin, and other collagen
5. Type I collagen (fibrillar collagen) is the most abundant in most connective collagen tissues. Collagen fibrils often are composed of more than one type of collagen.
6. Type IV collagen (basal lamina collagen) is a major component of the basal lamina and is a product of epithelial cells.
7. Collagen VII (anchoring-fibril collagen); the C-terminal ends associate to form dimers that subsequently are assembled into the anchoring fibrils that extend from the basal lamina into the underlying connective tissue.
8. Transmembrane collagen function in cell adhesion

Question 16

Microfilaments Assembly

Answer 16

1. Filamentous actin (F-actin) is an asymmetrical polymer. Two filaments form a helical microfilament. The plus end of a microfilament is the fast-growing end with net addition of subunits, whereas the minus end is the slow-growing end with net loss of subunits.
2. Three important actin-binding proteins (ABPs) that regulate microfilament dynamics are cofilin (which disassembles F-actin), profilin (which sequesters G-actin) and capping protein (which caps the plus end of filaments, inhibiting their disassembly)

Question 17

Actin Binding Proteins (ABPs)

Answer 17

1. Involved in bundling, capping, and membrane anchoring

Question 18

Actin and cell motility

Answer 18

1. Actin/microfilament are responsible for:
   
   1. sliding cytoskeletal fibers
   2. Moving cell membrane
   3. Moving cell organelles (intracellular transport)
   4. Moving cytoskeletal fibers
2. Carried out by actin and myosin working together
3. Microfilaments attach to the cell membranes via cell adhesion; i.e. anchoring complexes (focal contacts)
4. Focal adhesions serve as the mechanical linkages to the ECM, and as a biochemical signaling hub to concentrate and direct numerous signaling proteins at sites of integrin binding and clustering.

Question 19

Microtubule assembly

Answer 19

1. Tubulin synthesis is regulated by GTP whereas actin is ATP
2. Made up of tubulin dimers with + end and - end
3. Dynamic instability is a cycle of growth, catastrophe, shrinkage, and rescue and back to growth
   4.

Question 20

Microtubules

Answer 20

1. There are 9 dimers of microtubules (with two central singlet microtubules) in a cilium where as there are 8 triplets of microtubules in centriole.
2. Microtubules are also involved in intracellular transport
   
   1. Kinesin transports toward the Positive End
   2. Dynein transport toward the Negative End or toward the centrosomes.
3. Centrosomes consit of 2 centrioles consisting of mother and daughter centrioles
4. Centrosomes and microtubules play a key role in cell division

Question 21

Structural organization of collagen fibrils

Answer 21

1. Typical banded pattern of overlap (0.4D) and gap (0.6D) regions; approximately 1/4 stagger
2. Repeats every 300 nm

Question 22

Proteoglycans (or GAGs)

Answer 22

1. Proteoglycans are a large group of extracellular and cell surface–associated molecules that consist of a protein core to which glycosaminoglycan chains are attached.
2. Glycosaminoglycans are long chains of repeating disaccharide units consisting of a hexosamine and uronic acid.
3. The large number of carboxyl and sulfate groups in glycosaminoglycans makes them acidic (negatively charged). They readily bind various proteins and other molecules, and their hydrophilic nature allows them to bind large amounts of water.

Question 23

Glycoproteins

Answer 23

1. Glycoproteins play a role in collagen assembly; control the rate of assembly and fibril shape and diameter
2. Glycoproteins consits of a protein core and C- and N-linked glycans
   
   1. Tendons lacking proper glycoprotein structure lack regular shape and diameter
3. One of their primary functions is to bind cells to extracellular matrix elements.

Question 24

Collagen degradation

Answer 24

1. Collagen degradation is carried out by extracellular proteinases
2. fibroblasts also participate in the remodeling of connective tissues through the degradation of collagen and other extracellular matrix molecules and their replacement by newly synthesized molecules.
3. Two mechanisms for the degradation of collagen have been recognized: (1) the secretion by cells of enzymes that sequentially degrade collagen and other matrix molecules extracellularly, and (2) the selective ingestion of collagen fibrils by fibroblasts and their intracellular degradation (not really known).