Book Notes: ECM Part 2

• cell-cell junctions can send signals into the interior
• disintegrating the adherens jxns frees the adaptor catenins to wander around cytosol to spread a signal
• nonclassical cadherins may serve as co-receptors for signal receptors (i.e. VEGF is a survival signal that a receptor tyrosine kinase binds, but the receptor cannot bind the signal without VE-cadherin's help)
• selectins: cell-surface proteins that bind carbs (they are otherwise known as lectins)
• they also bind junctions, but specialize in temporary cell-cell adhesion
• example are white blood cells who patrol around and use this temporary binding to follow tracks so they can migrate between the bloodstream and tissue (i.e. mustering of white blood cells to location of inflammation)
• "rolling" motion along vessel endothelium
• transmembrane protein with lectin domain that binds specifically to oligosaccharides on another cell
• L-selectin: on white blood cells
• P-selectin: on blood platelets and inflamed endothelial cells
• E-selectin: on endothelial cells
• integrins: cell-surface proteins that bind other surface proteins
• also specialize in temporary cell-cell adhesion but is stronger than selectins
• work with selectins on white blood cells, but the stronger adhesion is necessary to overcome the selectins' affinity for blood vessel endothelium so that the white blood cell can escape from the vessel into tissue to save the day :D
• both selectins and integrins are heterophilic, they don't bind to selectins and integrins of the same type
• selectins bind glycoproteins and glycolipids
• integrins bind transmembrane immunoglobulins!
• immunoglobulin (Ig) superfamily: cell surface proteins with extracellular domains that are similar to antibodies
• intracellular cell adhesion molecule (ICAM): type of Ig bound by white blood cell integrin, heterophilic
• vascular cell adhesion molecule (VCAM): type of Ig bound by white blood cell integrin, heterophilic
• neural cell adhesion molecule (NCAM): type of Ig on neurons, homophilic, contribute to cell-type specific organization but not as necessary as cadherins
• synapse formation depends on axon finding the correct target tissue to form synapse with
• Ig adhesion molecules help by pairing up homotypically with target cells that express the same type of Ig
• example is Fasciclin3, who leads neuronal growth cones to connect with motor neurons in the muscle
• another example is Sidekicks, who matches neurons in different layers of the retina to growing axons
• synapse formation also requires complex assembly of receptors, ion channels, synaptic vesicles, docking proteins, etc, etc.
• scaffold protein: chains of proteins with various binding domains
• PDZ domains: about 70 A.A. long domains that bind cytosolic C-term tails of specific transmembrane proteins
• the various domains connect to the cadherins, channels, exo/endocytosis regulation protein, cytoskeleton, etc, etc
• multiple scaffold proteins can also unite to form network connecting all the components of a sypnase
• Discs large (Dlg): protein that assists in synapse formation, occluding jxn formation, control of cell polarity, and control of cell proliferation
• all epithelial cells (1) are polarized because one side faces basal lamina while the other faces the lumen and (2) form selectively permeable barrier by utilizing occluding jxns to prevent unfiltered leakage between cells
• tight jxn: the occluding jxn of vertebrates, prevent molecules from seeping past the cells in any direction and prevents membrane pumps from going to the wrong side (i.e. in gut lining, nutrients must go unidirectionally, from lumen to bloodstream, occluding jxn prevents them from leaking backwards and prevents nutrient pumps from going to the wrong side and pumping them backwards)
• experimentally show that tight jxns seal cell sheets by putting in a traceable molecule of a certain size
• visualization of molecule will show it never passes through the cell sheet
• sealing not COMPLETELY impermeable; small molecules can diffuse through
• but size limit of small molecules varies between tissues: bladder is almost totally impermeable to tiny sodium ions, while small intestine allows their passage
• sometimes cells loosen their junctions to allow nutrients to diffuse through easily, especially after meal, when the concentration gradient is high enough to have nutrients go in the correct direction by themselves: "paracellular transport"
• sealing strands: chains of adhesion proteins along the length of the cell close to the apex side that bind to partner chains on neighboring cells (not like one pocket or jxn like in cadherins, but chains of jxns all around the cell)
• claudins: the transmembrane adhesion proteins forming the sealing strands
• assisted by occulin and tricellulin, who seal the corners where 3 cells meet
• claudin family has members of varying structure to form different sized pockets to change the permeability
• paracellular pores: the selective channels formed by various combos of claudins
• occluding jxns have to occur above adherens and desmosomes, so all three are held together in junctional complexes
• Tight junction protein (TJP): a.k.a. ZO protein is a cytosolic scaffold protein that organize and anchor the adhesion proteins of the 3 types of jxns
• septate jxns: occluding jxns of invertebrates, very similar to vertebrate version
• cell-cell junctions and basal lamina govern the polarity
• experiment: grow MDCK cells suspended in gel all by itself, shows no polarity
• let them divide into small colony, the colony will organize itself into a vesicle shape, secrete basal lamina outside the vesicle, and all the cells now show polarity between the outside and inside of the vesicle
• polarization is spontaneous but depends on contact with neighboring cells
• experiment: screen for mutations in C. elegans that affect polarization of embryo
• PAR genes: family of genes that control polarization
• LKB1: vertebrate homolog of Par4, loss of LKB1 causes loss of polarization, overexpression of LKB1 causes self-polarization (not neighbor dependent), so cells can't orient TOGETHER into a sheet
• therefore, proteins assisting with polarity come in two categories: producing polarization in general, and orienting groups of cells correctly with landmarks like the basal lamina
• category 1: produce polarization in individual cell
• Par3: scaffold protein with PDZ domain, binds to the other two
• Par6: similar to Par3
• atypical protein kinase C (aPKC): binds to the two Pars
• the complex has binding sites for small GTPase Rac and Cdc42 and others
• Rac and Cdc42 control actin assembly (lack of Rac causes cells to polarize in the wrong direction)
• assembly of the complex is one specific part of the cell cortex recruits Rac and Cdc42 to polarize the cell towards that one part
• the complex also controls the Crumbs complex (with scaffolds Discs-lost and Stardust, assembles on apical side) and Scribble complex (with scaffolds Discs-large and Scribble, assembles on basal side)
• category 2: orient polarized cells to each other and to environment
• the proteins get coordinated to the different junctions by binding to tails of the transmembrane adhesion proteins
• from apical to basal:
• Crumbs complex
• tight jxn, Par3-Par6-aPKC complex
• Scribble complex
• Rac and friends induce cell to secrete basal lamina ECM components to opposite end of cell (since most of the proteins were associated near the apical side with the tight jxns, the "opposite" side is basal lamina)
• planar cell polarity: cells in a tissue with apico-basal polarity show additional polarity towards another direction perpendicular to the apico-basal axis.
• example: in trachea, all cells are lined from apical to basal side horizontally, BUT their cilia must sweep in a vertical direction to shift mucus and dust upwards and out
• studying mutations revealed important proteins whose function is not exactly known yet (they are also oddly named)
• Frizzled and Dishevelled (these two part of a signal pathway called "Wnt"), Flamingo and Dachsous (these two code for cadherins)
• gap junction: channel between cells that allow them to share cytoplasm without having to exocytose stuff and the other cell endocytose it (plant counterpart is plasmodesmata)
• channel is about 2-4 nm long formed by connexins and innexins.
• ions and small molecules flow easily through them (dyes and electrical currents transfer quickly throughout a sheet of cells)
• channel is about 1.5 nm wide (ions, sugars, A.A, N.T., vitamins, cyclic AMP and other second messengers) but can't share macromolecules (proteins, DNA, etc)
• connexon: a hemichannel formed by six connexins (4 pass transmembrane protein)
• the two connexons of two cells align and stick together to form whole channel
• gap junction consists of a lot of connexons, so it looks more like a sieve of pores rather than one giant channel
• connexons can be assembled out of combos of different kinds of connexins to produce different permeabilities
• even two hemichannels can be made of radically different connexins and still connect together
• connexin have half life of a few hours, connexons are always added around existing gap jxns and removed from center of existing gap jxns
• connexins are inserted into membranes and delivered to the plasma membrane by exocytosis
• they diffuse around the plasma membrane until they hit an existing gap jxn and get trapped there
• unpaired hemichannels are either closed or can be opened to serve as channels for release or entrance of small molecules
• gap jxns enable instantaneous transfer of action potential between joined cells
• useful in nerve cells where speed and synchrony is important in the response (i.e. escaping from surprise attack)
• useful also in maintaining homeostasis of a tissue (small molecule concentration can fluctuate from cell to cell, but having that channel allows them to diffuse out of high concentration cells to boost low concentration cells)
• even whole channels can flip between open and closed, and permeability is reduced by low pH or high calcium concentration
• ECM is full of calcium, so if one cell is punctured, calcium rushes in, the gap junction senses this and closes, protecting neighbors from the damaged cell (remaining connected means whatever gets into the puncture cell can also get into the healthy cells)
• plasmodesmata: the only jxn for plants, is a pore in which the plasma membranes of different cells are CONNECTED, not just connection by a protein channel
• channel about 20-40 nm diameter
• desmotubule: narrow cylindrical tubule that connects to the ER in the center of the channel
• cytosol is shared around the desmotubule
• basement membrane (basal lamina): thin, tough, but flexible sheet of ECM that is essential to all epithelia
• 40-120 nm thick, surrounds muscle and fat and epithelial cells
• function in forming structure, filtering between its two sides, influence metabolism, promote cell survival, proliferation/differentiation, act as pathway for migratory cells
• skin depends on connection the basal lamina which holds it fast to the underlying dermis
• basal lamina made of FIBROUS PROTEINS (glycoproteins with short oligosaccharides) and GLYCOSAMINOGLYCANS (GAGs, linked to proteins to make proteoglycans)
• lamina composition varies, but there are key components
• glycoproteins: laminin, type IV collagen, nidogen
• proteoglycan: perlecan
• associate with other molecules: collagen XVIII, fibronectin
• laminin-1: classical laminin, has 3 polypeptide chains arraned like bunch of flowers in a vase
• exist various isoforms of the each chain type, so laminins come in various combos like connexons
• interact with each other at heads to form a sheet (PRIMARY ORGANIZER)
• type IV collagen: 3 long protein chains that form superhelix, interrupted in several places to allow bending
• interact with each other at terminal domains to form flexible but strong network (TENSILE STRENGTH)
• laminin binds to perlecan and nidogen and cell laminin receptors, while collagen binds to perlecan and nidogen
• so perlecan and nidogen connect the networks of laminin and collagens (LINKERS)
• cell receptors like dystroglycan grab laminin by their feet, laminin heads network together to form a sheet, nidogen and perlecan link the laminin to collagen network
• examples of diverse functions of basal lamina
• kidney: superthick lamina blocks maromolecules from leaving blood when filtering for urine
• epithelium movement: blocks fibroblasts of connective tissue from touching epithelial cells, but allows passage by macrophages and lymphocytes (they have proteases to cut holes through the lamina for them)
• regeneration: studied at neuromuscular jxn where nerve meets muscle, but they are separated by basal lamina
• lamina has special isoforms of all of its components
• when the nerve cell happens to be destroyed, new axons locate the old site by recognizing the special markers of the basal lamina that still marks the junction
• works similarly for muscle cell regeneration
• nerve cell deposits agrin proteoglycan at the lamina site, who induces muscle cells to form acetylcholine receptors on their surface, while muscle cells deposit special laminins that bind to voltage gated ion channels of nerve cells
• when either or both nerve and muscle gets destroyed, muscle regenerates first by natural wound response, but gets positioned correctly by the existing agrin, then growing axons come down to find the old site by binding to the existing laminins

pg. 1145 - 1169
A.A. = amino acids
N.T. = nucleotides