Lecture 3+4 How Do Cells Communicate.pdf

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University of Toronto St. George
Cell and Systems Biology
William Navarre

CSB328H1 © Lisa | Page 13 L E C T U R E 3 & 4 : C E L L - C E L L C O M M U N I C A T I O N I N D E V E L O P M E N T Reading: Gilbert Chapter 3, page 136 (in Chapter 4)  morphogenesis: the formation of organized form CELL ADHESION (ectodermal) DIFFERENTIAL CELL AFFINITY  E.E. Just – cell types have diff cell membrane components  these diff components enables formation of organs  Townes & Holtfreter (1955) – discovered segregation of cell types after dissociation  prepared single-celled suspensions of each germ layer & combined them  cells were separated in alkaline solns for separation  when cells of diff germ layers were combined, & pH normalized, cells stuck together  they combined single-cell suspensions in various ways 1. found that reaggregated cells spatially segregate (to its own region, Fig 3.1) 2. found that the final positions of the reaggregated cells reflect their positions in the embryo – selective affinity (Fig 3.2)  (C) endoderm separates from ectoderm & mesoderm, and is enveloped  selective affinity: o inner surface of ectoderm has +ve affinity for mesodermal cells -ve affinity for endodermal cells o mesoderm has +ve affinity for ectodermal AND endodermal cells Selective Affinity of Cells Germ layer Ectoderm Mesoderm Endoderm 3. selective affinities change during dvpmt Ectoderm + –  cells interact diff w each other at diff times Mesoderm + + Endoderm – +  segregation of tissue cells (w trypsin) from later-stage animals reaggregated to form tissue-like arrangements (Moscona 1961; Giudice & Just 1962) Page 2|CSB328H1 © Lisa Zhao2013 THE THERMODYNAMIC MODEL OF CELL INTERACTIONS  forces direct cell mvmt (what forces?)  Malcom Steinberg (1964) – the differential adhesion hypothesis (cell types differ in their strength of adhesion) 1. cells migrate centrally/peripherally to diff cell types  Fig 3.3 (A) 5h, randomly arranged cells, (B) 19h, only neural retina cells on periphery (C) 2 days, 2 distinct layers: pigmented retina cells central to neural retina cells (scattered cells – dead) 2. cell interactions form a hierarchy: if cell type A is internal to B & C is internal to C, then A will always be internal to C 3. cells interact to form an aggregate w the smallest interfacial free NRG (most thermodynamically stable)  A-A > A-B/B-B = A cells central  A-A ≤ A-B = random mix  A-A >> A-B = (no adhesivity twds one another) separate aggregates of A & B  early embryo is in equilibrium until gene activity changes surface molecules o resulting mvmts seek to restore equilibrium  therefore, differing strengths of adhesion  cell sorting  cell types w greater surface cohesion (surface tension, strength of adhesion) migrate centrally (Fig 3.4)  diff types of adhesion molecules causes thermodynamic differences (Moscona 1974) CADHERINS & CELL ADHESION  cell adhesion molecules mediate cell-cell adhesions (varying #’s & types)  main cell adhesion molecule: cadherin (calcium-dependent adhesion)  Takeichi (1987) – cadherins are crucial for spatial segregation of cell types & organization of animal form  inactivation of cadherin (synthesis, or directly) prevents formation of epithelial tissues & cause cell disaggregation (Fig 3.5B)  cadherins are transmembrane proteins (Fig 3.5A)  interact w cadherins on adjacent cells  anchored inside cell by catenins  catenins attached to actin microfilament (cytoskeleton) = mechanical unit  cadherin-catenin complex = adherens junction (hold epithelial cells together)  functions: 1. adhere cells together 2. link & assemble actin cytoskeleton (provide mechanical force for forming sheets & tubes) 3. signal molecule (change gene expression)  types:  E-cadherin – all early mammalian embryonic cells, epithelial tissues of later embryos & adults  P-cadherin – placenta (attachment to uterus)  N-cadherin – cells of the developing CNS, mediate neural signals  R-cadherin – retina formation  protocadherins – lack attachment to actin  expression of similar protocadherins keeps migrating epithelial cells together  expression of diff protocadherins helps separate tissue  strength of cadherin interactions determines cell surface tension & adhesivity  determined by # (quantitative) OR type (diff cadherins bind to each other, qualitative) CSB328H1 © Lis| Page 3013  Steinberg & Takeichi (1994) – quantitative cadherin-dependent cell sorting  when cells expressing diff # of P-cadherins put together, those cells w more had higher surface cohesion & migrated internally  Foty & Steinberg (2005) – surface tension ∝ cadherin # (linear relationship, Fig 3.6)  also, actin organize cadherins in forming stable linkages bw cell Quantitative Qualitative  Duguay et al. (2003) – R-cadherin & B-cadherin don’t bind well (qualitative)  N-cadherin imp in separating precursors of neural cells from precursors of epidermal cells  E-cadherin present in all early embryonic cells  cells to become neural tube lose E- and gain N-cadherin  Kintner et al. (1992) – epidermal cells expressing N-cadherin OR blocked synthesis of N- cadherin)  no border bw skin & nervous system (Fig 3.7E)  Oberlender & Tuan (1994) – timing of some dvpmtal events depends on cadherin expression  N-cadherin appears in the mesenchymal cells of chick embryo leg right before these cells condense to form cartilage  it is not seen before or after condensation  blocked N-cadherin right before condensation  no condensation of mesenchyme cells  therefore, signal to begin cartilage formation in chick limb is appearance of N-cadherin  cadherins work with other adhesion systems – ex. implantation of mammalian embryo  first differentiation of mammalian embryos distinguish trophoblast cells  trophoblast cells express many adhesion molecules to adhere to uterine wall o E- & P-cadherins recognize similar cadherins on uterine wall (Kadokawa et al. [1989]) o receptors (integrin proteins) for collagen & herapran sulfate glycoproteins on uterine wall Page 4|CSB328H1 © Lisa Zhao2013 Importance of Cadherin in Cell Adhesion & Morphogenetic Movements  cadherins & the actin cytoskeleton mediate epithelial cell shape change into sheets/tubes  proteins that mediate cadherin-dependent remodeling of the cytoskeleton: 1. myosin II (non-muscle myosin) 2. GTPases (Rho family) – convert soluble actin into fibrous cables that anchor at the cadherins  neural tube formation (vertebrates) & internalization of mesoderm (Drosophila) require migration of cells from out to in  neural tube formation in vertebrates – involution of the frog neural tube  all gastrula cell membranes have C-cadherin  presumptive neural tube ectoderm cells also contain N-cadherin in apical (upper) region  presumptive epidermal cells of ectoderm also contain E-cadherin on lateral & basal surfaces  actin in apical region of neural cells causes them to change shape & enter embryo to form neural tube  actin on lateral sides of epidermal cells enable the migratory mvmts of the epidermal cells over the surface of the embryo  removal of N-cadherin results in failure of actin to assemble apically  no neurulation (Fig 3.8, non-functioning N-cadherin gene on left side, uninjected right side develops normally)  internalization of the mesoderm in Drosophila  ventral epithelial cells form a furrow (via apical constriction) & migrate inside the embryo (Fig 3.9)  apical constriction results from rearrangement of actin & myosin II to the apical end of cell  brought about by the Twist gene, expressed only in nuclei of the most ventral cells (Fig 3.10A)  buildup of actin accomplished by the binding of a Rho GTPase & β-catenin to E-cadherin on the apical portion of the membrane in the most ventral cells (Fig 3.10B)  actin-myosin complex in apical cortex constricts like a drawstring, cells change shape, buckle inward, & enter embryo  forms mesoderm Fig 3.10 (A) Ventral cells are defined by the expression of transcription factors Twist & Snail. These cells accumulate myosin II at their apical surfaces. When myosin interacts w actin already present, the cells begin to constrict apically & thus invaginate. (B) Before the initiation of ventral furrow formation, Rho GTPase & β-catenin both reside along the basal surface (facing the interior of the embryo) of the ventral cells. β-catenin is also found in a subapical region in all cells. Formation of the ventral furrow begins w the relocalization of Rho & β-catenin, which move from the basal surface to accumulate apically, at the opposite end of the cell. CSB328H1 © Lis| Page 5013  insect (Drosophila) trachea formation  tracheal (respiratory) system forms form epithelial sacs composed of cells that become reorganized into 1°, 2°, & 3° branches  instructions for folding come from outside the cells – Branchless (Bnl) protein secretion by nearby cells  Bnl is a chemoattractant (soluble molecules that attract cells to migrate along an increasing conc gradient towards the cells secreting the factor)  Bnl binds to a receptor on epithelial cell membranes which activates Breathless (Btl) protein that induce migration of the leader cells & tube formation (Fig 3.11)  the cells receiving the most Bnl lead the rest o migrates by changing shape – rearranges its actin-myosin cytoskeleton via a Rho GTPase-mediated process  followers (connected by cadherins) receive a signal from the leading cells to form the tracheal tube  3° cell migrations of the dorsalmost secondary branches of the sacs along grooves bw muscles cause the trachea to become segmented around the musculature – close interaction (Fig 3.11B) Page 6|CSB328H1 © Lisa Zhao2013 CELL MIGRATION  involves abundant reorganization of the actin cytoskeleton  in epithelia, the cells at the edge provide the motive force (other cells passively follow)  in mesenchymal cell migration, indiv cells become polarized  4 stages of cell migration: 1. polarization – cell defines its front & back (Fig 3.12A)  by diffusing signals (ex. chemotactic factors) or ECM signals  signals reorganize cytoskeleton so that front is diff from back 2. protrusion of the leading edge  polymerization of actin provides the mechanical force  creates long parallel bundles (forming filopodia) or broad sheets (forming lamellipodia, Fig 3.12B)  membrane-bound Rho GTPase activates the WASP-N proteins to nucleate actin & connect it to cadherins & cell membrane 3. adhesion to ECM  cell needs to push off of something – attaches to surrounding ECM  process mediated by integrins – span the cell membrane, connects the ECM to actin  connections of actin to integrin form focal adhesion sites on the cell membrane where the membrane contacts the ECM  myosin provide the motive force along these actin microfilaments, & they are linked w the lamellipodial actin at the sites of adhesion 4. release of rear adhesions  stretch-sensitive Ca channels open & Ca ions activate proteases that destroy the focal adhesion sites CELL SIGNALLING INDUCTION & COMPETENCE  cell behaviours are regulated by signals received from another cell – induction (interaction at close range bw 2 or more cells or tissues)  the 2 components of inductive interactions: 1. inducer – the tissue that produces the signal(s)  usu a paracrine factor (protein) – secreted into the extracellular space, alters the behaviour/differentiation of adjacent cells (close by) 2. responder – cell/tissue being induced  must have: 1) a receptor protein for the inducing factor 2) the ability to respond to the signal – competence  vertebrate eye formation  paired regions of the brain (optic vesicles) bulge out & approach the surface ectoderm of the head where they release paracrine factors  only the head ectoderm is competent to respond & are induced to form the lens of the eye (Fig 3.13)  genes for lens proteins are induced & expressed in the head ectoderm cells  Rho-family GTPases are activated to control the elongation & curvature of the lens fibres  the prospective lens cells secrete paracrine factors that instruct the optic vesicle to form the retina (the lens & retina co-construct each other – reciprocal paracrine interactions)  usu, one induction will give a tissue the competence to respond to another inducer CSB328H1 © Lisa| Page 713  ex. vertebrate eye induction 1. inducers from the foregut endoderm & heart-forming mesoderm 2. inducers from the anterior neural plate – promotes the synthesis of Pax6 (anterior ectoderm transcription factor) o Pax6 provides the competence for the ectoderm to respond to optic cup inducers 3. optic vesicle secretes 2 paracrine factors: 1) BMP4 – induces production of Sox transcription factor 2) Fgf8 – induces appearance of the L-Maf transcription factor  lens formation & activation of lens-specific genes requires Pax6 + Sox2 + L-Maf in the ectoderm RECIPROCAL INDUCTION  reciprocal induction: an induced tissue can induce other tissues, including its own inducer  a structure doesn’t need to be fully differentiated to have a function (induce)  ex. the forming lens in turn induces the optic vesicle – the inducer becomes induced  optic vesicle  optic cup  pigmented retina & neural retina (2 layers) Instructive & Permissive Interactions  Howard Holtzer (1968) – 2 major modes of inductive interaction: 1. instructive interaction – a signal from the inducing cell is necessary for initiating new gene expression in the responding cell  ex. optic vesicle induces ectoderm to form lens 2. permissive interaction – the responding tissue has already been specified & needs only an envmt that allows the expression of these traits  ex. ECM doesn’t alter the cell type, but enables expression EPITHELIAL-MESENCHYMAL INTERACTIONS Regional Specificity of Induction  2 main tissues of skin:  outer epidermis (epithelial tissue derived from ectoderm)  dermis (mesenchymal tissue derived from mesoderm)  chick epidermis signals underlying dermal cells to condense  condensed dermal mesenchyme responds by secreting factors that cause the epidermis to form regionally specific cutaneous structures (Fig 3.17)  recombination of separated epithelium & mesenchyme – the same epithelium develops cutaneous structures according to the region from which the mesenchyme was taken  mesenchyme plays an instructive role to diff genes in the responding epithelial cells Genetic Specificity of Induction  mesenchyme gives instructions on which genes to activate, but epithelium can only respond w what its genome permits  Spemann & Schotte (1932) – interspecific induction: newt & frog ectoderm transplant  flank ectoderm from early frog gastrula to oral ectoderm area of newt gastrula & vice versa (Fig 3.18)  produced chimeras: salamander larvae w froglike mouth & tadpole w salamander teeth  mesenchymal cells instructed the ectoderm to make a mouth, but the ectoderm made the only kind of mouth it “knew” how to make Page 8|CSB328H1 © Lisa Zhao2013  instructions sent by mesenchymal tissue can cross species barriers – organ-type specificity (ex. feather or claw) is controlled by the mesenchyme  but response of the epithelium is species-specific – species specificity is controlled by the responding epithelium PARACRINE FACTORS: INDUCER MOLECULES  juxtacrine interaction: interaction of membrane proteins on one cell surface w other cell surfaces  paracrine interaction: interaction bw a cell & a protein synthesized by a neighbouring cell  autocrine interaction: cells respond to the paracrine factors they secrete  proliferation SIGNAL TRANSDUCTION CASCADES: THE RESPONSE TO INDUCERS  many of the same paracrine factors are used to induce dvpmt of diff organs  many factors are conserved throughout the animal kingdom  4 major families of paracrine factors: (based on structure) 1. fibroblast growth factor (FGF) family 2. Hedgehog family 3. Wnt family 4. TGF-β superfamily –bone morphogenic proteins (BMPs), Nodal proteins  signal transduction cascades: pathways of response whereby paracrine factors bind to a receptor that initiates enzymatic rxns that result in either regulation of transcription factors (gene regulation) or regulation of the cytoskeleton (shape change or migration)  structure & function of a receptor tyrosine ki
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