lecture 14 Flashcards
what are examples of tissue morphogenesis
epithelial tube formation, branching morphogenesis, convergent extension and axis elongation
what is tissue morphogenesis controlled by
combo of intracellular signaling, proliferation, and active forces
what does intracellular signaling control
when and where these forces are created within the tissue
what is the biggest factor in tissue morphogenesis
active forces
what are active forces
forces produced by the material itself to change its own shape
why do we call embryonic tissue active material
because it can change its own shape during developmente
what two things happen in tube formation/convergent extension
flat surface of embryo invaginates and deepens to form primordial spinal cord/neural tube
what else happens
embryo elongates at one axis
when is this invagination/elongation process happening
as we go from gastrula stage to neurula stage
describe elongation
tube begins to form; embryo begins to get long at one axis and shorter in another axis
what is convergence
cells are coming together in one direction and elongating in another direction
what is convergent extension
flow of material towards the tube, towards the middle as it begins to elongate in perpendicular direction
what do many organs undergo during development
branching morphogenesis
describe branching morphogenesis in salivary glands
goes from embryonic nub, through cell divisions + clefting & branching, get grape like clusters of epithelial cells that secrete saliva down ducts into oral cavity
what do salivary glands undergo
rounds of clefting and branching –> go from single bud to a elaborate, highly branched epithelia
describe branching morphogenesis
branching splits a little, widens and forms a discrete cluster (we get distance b/w adjacent branches), repeats again and again to get smaller clusters of epithelial cells
what does basememt membrane separate
separates epithelial cells from region outside of organ
does basement membrane maintain its barrier while epithelial cells do their thing
yea duh
what is critical for organ development
branching morphogenesis
what is formed thru branching morphogenesis
salivary glads, organs like kidneys, lungs, blood vessels
what shapes tissues
physical forces
how do we measure physical forces in tissues
laser ablation, dual micropipette aspiration, traction force microscopy, atomic force microscopy
what does laser ablation measure
measures tension along cell-cell junctions thru recoil velocity
what is tension in a tissue reflective of
contractile activity within the cells making up that tissue
what can you do through cadherin junctions
trigger positive feedback loop to get a massive amount of actin contractility applied to junctions
how can we detect a cell that’s hella contractile or tense vs. one that isn’t
look at cells thru a microscope, label cadherin
what next (laser ablation)
use laser to break connections b/w cells; more tension cell is under, more it recoils
what happens if you cut connections in a cell w/ no tension
it doesn’t move
give an example of cell that’s under a lot of tension
developing drosophila embryo under hella tension
what does dual micropipette aspiration do
measures strength of cell-cell junctions
what is another relevant physical force
strength of adhesion; how strong are two cells bound to each other
what does strength of adhesion reflect
actin contractility
what does number of cadherins interacting w/ each other correlate w/
stronger adhesion between cells
how do we see how strong cells are sticking together
move cells around, let them stick and adhere
what is indirect way of measuring adhesion strength
how far you have to move the pipettes before you break the bonds
what does traction force microscopy do
measures force generated by cell-matrix adhesions (tensile stress)
what are traction forces
forces that strengthen and grow adhesions
what do traction forces do / help with
apply to extracellular environment, help pull the cell forward or remodel the extracellular environment
what else do traction forces do
align collagen fibers; or in wounds grab onto fibronectin that’s spilled out of blood serum, unfold it to reveal additional binding sites to promote adhesion during a wound healing response
how do we measure traction forces
flexible pillars; measure how much pillars deflect
describe how we measure traction forces
plate cells on flexible gell, put small beads, let cells attach & adhere to gel and apply traction forces to it
what is the distance the beads move when you kill the traction forces related to
how high those traction for es were
what happens if gel is being stretched by an enormous amount
beads are gonna move greater distance as the gel relaxes
basically what is it
beads move cuz of traction forces; stop the traction forces, beads will relax and go back to where they were before
what is atomic force microscopy
measures the material properties of individual cells w/ functionalized probe
what is another characteristic of cells
whether they are soft or stiff
what are soft cells
highly metastatic cells
what cells are stiff
normal epithelial cells
describe atomic force microscopy
use small probes –> how much force microscope has to apply to deform the cell is directly related to how stiff the cell is
what happens if the cell has to apply a lot of force to dent the cell
it would be stiff
what happens if the cell doesn’t apply as much force to dent it
cellis soft
what changes in a cell to cause it to become rigid or stiff
pressure & rigid membrane / cytoskeleton
what is the final way to measure forces
pressure
describe micropressure system
stick needle into pressurized compartment
what happens to pressure as cells move
pressure goes up and down when they are migrating vs. non-migrating
where do forces need to be generated to cause a planar field of cells to become invaginated (to form neural tube)
exactly where the tube is going to form
describe how the force is applied
not uniform; it’s a gradient, most strong in the center and drops off towards the edges
what is another example of how forces can participate in developmental processes
traction forces
what is important in epithelial tube formation
cell-cell junction force generation; cadherins, alpha catenin unfolding to activate force where the tube is gonna form
what is an example of how traction forces can be used to create tissue (tendon)
collagen; type 1
what happens during development when a tendon needs to form
there’s a lot of collagen deposited
how is collagen arranged
randomly in embryonic tissue
what happens when an embryonic fibroblast gets in the right area
it’s able to begin to remodel the matrix
what is collagen remodeling done in response to
signaling, growth factor, physical environment it’s embedded in
what does extracellular matrix act as
blueprint/scaffold to create the tissue
what is next step in tendon formation
embryonic fibroblasts remodel the matrix to form that blueprint (where traction forces come in)
what happens when you add a cell to this otherwise randomly arrayed collagen fibers
become less uniform
what is step 2 of developing tendon
cells align the collagen matrix in addition to depositing new matrix
what does aligned matrix do
forms scaffold for additional cells to come in, deposit more matrix, ultimately forming a tendon
what is the key point
traction forces align the randomly deposited matrix at earlier step of development to form blueprint for where a tendon is ultimately gonna form to attach the muscle to bone
how are stress fibers created to generate the traction forces
integrins are receptors that interact w/ collagen;
where are integrins
on cytoplasmic face of membrane they’re connected to actomyosin thru talin & vinculin
how do we get more contractility
positive feedback can be triggered from outside or inside to unfold talin, get bigger, stronger adhesions generating more traction forces
what does tissue remodeling depend on
coordination of actin-mediated contraction w/ cell-cell adhesion
when we talk about cell-cell junctions what are we talking about
bundles of actomyosin filaments near apical surface of epithelial cells (on sides)
when we talk about contractility that shapes epithelial cells
we talk about bundle of actin filaments across epithelial cells that are connected at cadherins
what do adherens junctions do
respond to forces generated by actin cytoskeleton
describe how cadherins bind to each other
cadherins homophilic binding to the partner cadherin on adjacent cell
how are cadherins plugged into actomyosin filaments
through alpha catenin (NOT talin)
what is alpha catenin
the force sensor
what happens if something were to pull on the cell or from inside the cell on the adhesion
alpha catenin would unfold, provide additional binding sites for vinculin, bring additional actomyosin fibers strengthening the adhesions junctions and increase tension in that part of the tissue
how are those stress fibers created
formins
what are formins activated by
rhoA which nucleates long straight actin filaments that will be bundled into stress fibers
what is the cross linker
alpha actinin
how does myosin enter
rhoA is activated, activates ROCK/MLCK, phosphorylates myosin regulatory chains in neck region of myosin heavy chain –>binds actin, unfolds tail so it does bipolar filaments that slip into bundled by alpha actinin
how does myosin generate force
myosin conformational cycle driven by ATP hydrolysis release and binding
when we talk about branched actin network, what does it mean
Rac1 arp 2/3, 70* angles etc.
what is needed in epithelial tube formation
something has to happen contractility wise to get that initial invagination, coupled with proliferation & other forces that get epithelial tube formed
where is belt of actomyosin fibers located
towards apical surface (top of epithelial cells but still on the sides)
how is belt arranged
continuous belt along the perimeter of the epithelial cell
what happens when it becomes activated
everything contracts uniformly around the perimeter (purse-string mechanism), cell becomes narrower overallw
is the contractility uniform ALL around the cell?
no, other wise whole cell would shrinkw
where is this contractiion
near the top; top gets narrower while the bottom stays relatively wide
what does apical constriction lead to
tube formation and branching morphogenesis
describe drawstring mechanism
top gets narrow, bottom stays relatively wide
what is consequence of drawstring mechanism
narrower top, wider bottom –> exactly what it takes to get it deformed from flat to that first invagination
what does purse string mechanism / apical constriction lead to
triggers a domino effect to get the full tube formation
describe the shape of it
cone-like shape
what does this process depend on
cadherin cell-cell junctions (connected to actomyosin filaments in a full belt along the perimeter of the cell (interconnected in adjacent cells)
what happens when cells contract
you get non-uniform change in shape that leads to first invagination and first step on the way to epithelial tube formation
what does tube formation require
proliferation and actin-mediated contraction
how does that contractility get activated in that local zone? what changes in cells at cell-cell junctions to get the cells to become activated
(local) paracrine signaling activates rhoA, rhoA activates actomyosin contractility by applying a bit of force (increasing myosin a little bit), unfolds alpha catenin to get positive feedback loop
what happens after positive feedback loop
all of a sudden cells are contracting at their apical surfaces, entire tissue is invaginating in that one little strip
what does this process lead to
epithelial tube formation
describe rhoA activity of other parts of embryo
lower rhoA activity
what does convergent extension do
elongates developing tissues
what is convergent extension
what happens as the embryo begins to deform form a round spherical object to a lengthening embryo
where does convergent extension happen
all over the region of cell that’s converging and extending
how does convergent extension lead to elongation of tissues
hundreds, thousands of cells in tissue are doing this continuously leading to flow in one direction and extension in the other
what happens to blue cells
come together and form a connection
what happens to green cells
get pushed apart in the opposite, perpendicular direction
what happens to entire embryo as this occurs
shrinks top and bottom, elongates left and right
what do cells do
come together to and bottom, go apart left and right
what is the net result of convergent extension
macroscopic change to embryo where it begins to lengthen dramatically in one axis
where does molecular machinery come into play
cell-cell junctions
what happens to one set of cell-cell junctions
goes away, as the other set is being created
why is one set of cell-cell junctions eliminated
to allow blue cells to be able to make contact w/ each other
what is the final step
they form a new cell cell junction
what happens to cell junctions
junction between gray cells is dissolved, junction between green cells forms
what is end result
tissue shrinks in one axis, elongates in perpendicular axis
what are the molecular mechanisms that drive this process
actomyosin contractility that begins to put tension on the junctions that need to go away, draw those cells together
what else is it coupled with
endocytosis
what happens as those junctions go away
cadherins and membranes are recycled into the cell, allows cell cell junctions to be erased
what happens when Rac1 gets activated to expand the zone of contact
rhoA gets activated to strengthen and grow the cell-cell junctions
what is final piece of the puzzle
as you’re getting cell-cell junctions formed, you’re gonna have exocytosis of cadherins up to the cell membrane to help form new cell-cell junctions
what is overall process of convergent extension
junctions between cells go away
what happens for these junctions to go away
actomyosin contractility is increased, and E-cadherin is endocytosed away from the cell
how is contractility increased
downstream of RhoA, activates myosin 2, bipolar filament assembly, additional actomyosin filaments, etc.
how is it endocytosed
clathrin-mediated endocytosis (clathrin coats, uncoats, rab effectors, v snares, t snares, etc.)w
when is e-cadherin exocytosed
first step of convergent extension
what happens as a result of endocytosis and contraction
cell-cell junctions shrink and other cells get closer and closer together
how do they form cell-cell contacts
rac-dependent expansion, rho-dependent strengthening, secretion of E-cadherin to those newly formed junctions
examples of tissue morphogenesis
tube formation, convergent-extension, branching
what are significant factors in shaping developing tissues
physical forces (cell proliferation, material properties, active forces (pressure and contractility))
how does tissue shaping/morphogenesis occur
signaling and physical forces work together
