Unit 10 - Cell Motility

Types of Cell Motility

Actin-dependent migration

Actin Dependent Migration

Cell Signaling

signals from outside - chemical, neighbor cells, mechanics, electrical, photo; responses inside the cell (change in cell phenotype) - trigger new gene expression, cel movement or shape change, cell cycle, or apoptosis

Movements of the cell

driven by the cytoplasm and associated motor proteins - ex. migratory frog cell expressing EGFP-CLIP1, a live-cell reporter of MT dynamics; can also be corrdinated and is responsible for buidling structures and organs - ex. migration of epithelial cell cluster produces the sensory lateral line in fish (sends out a migratory cluster of stuff down the line)

Why we don’t always use human cells?

1. ethics

2. experimental design

3. time

4. money

Why do we use yeast cells?

function of a protein in yeast is the same in us; cheap, abundant, and reproduce rapidly; form distinctive polarized structures under defined conditions (“on-demand”) and can be synchronized

Why are yeast cells not useful?

because they can’y make multicellular organisms

Cell Motility - Cell Shape and Locomotion

cell polarization (in yeast cells → cells want to polarize), RNA localization (putting proteins where it wants), plasma membrane protrusion, external signals guide cell migration, cell ex. keratocytes (good migration cells) and neurons;

Cell Motility - Cell Needs

to sense cues from environment, ability to directionally organize and polarize their cytoskeleton (all about location), generate physical force to move, persistence to keep “on-track”

Involves a coordinated deployment of components and processes of the cytoskeleton

cell polarization, shape change, and motility involves a coordinated deployment of components and processes of the cytoskeleton - dynamic assembly and dissaembly of polymers (non covalent bonds), regulation and modification of their structure by associated proteins, and actions of motor porteins among the polymers

Yeast: Saccharomyces cerevisiae

single cell eukaryote; type of fungus with a chitin cell wall; complex life cycle with asexual and sexual reproduction; can be haploid or diploid - budding; conjugation - a and alpga haploid cells fuse; most have human homologs (can use them to understand human protein-protein interactions); can polarize by conjugation/shmooing or cell division/budding

Conjugation/schmooing

polarized behavior during conjugation; each haploid cell can secrete a diffusible mating factor (signal) which activates a pathway that triggers the cell to produce a “polarized” response; 2 types - a and alpha; ex. a extends a protrusion in response to an alpha mating factor

Cell Division/Budding

polarized assembly of a new daughter cell during cell division; creates 2 cells - mother and daughter; mother selects site for new daughter cell next to old bud scar and directs protein complexes to that site; steps - cel cycle, mitosis, and cytokinesis; daughter cell is different than mother cell - yeast wants to put things (proteins and different genes that are on/off) in the daughter cell (differential transport of ash1 mRNA → daughter recieves ash1 and mother is depleted of ash1) ; generates a “polarized” response to an internal stimulus

Polarity Generation in other cells

animal cell suse extrernal stimuli (chemoattractants) to direct a complex response - neutrophils are immune cells that polarize to track invasive pathogens; keratocytes are epidermal cells from fish/amphibian skin and are useful for studying cell motility because they are big and fast; extrernal cues can guide cell migration - chemotaxis, haptotaxis, durotaxis, and galvanotaxis

Chemotaxis

cells follow gradients of diffusible factors

Haptotaxis

cells track immobilized molecules (Hansel and Gretel)

Durotaxis

cells follow gradients in substrate stiffness (soft vs stiff in places)

Galvanotaxis

cells guided by applied voltage (follow voltage gradient low → high)

Disadvantages of a pyrene actin assay

it is an average (bulk assay)

TIRF

total internal reflection fluorescence

Polarity

input layer - intracellular (inside) and signaling (outside); core processes can be polarized - universal processes (Rho/Rac/Cdc42); must occur at the output layer (most important part about cell motility - intracellar - ex. Ameboid motility - branched actin at front and actomyosin contraction at rear

Rac

front of cell; has a direct interaction with PI 3-kinase, PI(4)P and PAK and indirect with WASp family; causes decreased myosin activity so less stress fiber formation; causes branches actin web in lamellipodia (capping protein, ARP, and filamen)

Problem about cells

come in a variety of shapes and use different strategies for migration but they do share a common set of molecular machines for all steps of polarization and movement

Universal Principles of Cell Motility

F-actin assembly and myosin contractility controlled by G-proteins; there are numerous downstream effectors of Rac (front of cell) and Rho (back of cell); these are biomolecular switches;

Rho

direct interaction with Rho kinase (ROCK); formins control actin bundle growth; more stress fibers and integrin clustering and focal adhesion formation

Universal Principle of Cell Motility

assembly of branched F-actin at the leading edge; actin polymerization drives the membrane forward

the process at a neutrophil changing direction as it hunts bacteria is most like what event is the yeasts’s life

conjugation/schmooing

Neurons vs Fibroblasts

actin - cell cortex and allows extension and growth via “growth cone”; microtubules - oriented asssembly in axon to deliver vesicles to synapse; intermediate filaments - neurofilaments hold the axon together into compact structure; nuerons - send out processes to connect (dendrites - short - receive signals - associated with yje cell body and axons - long - transmit signals)

Growth Cones

how neurons connect to other cells; they leave the axons or dendrites to connect to the neuron cell body left behind; migrate according to tropic cues; neuron version of the front end of a migratory cell; steps - filopedia explore micro-environment → cells bind and modify diverse signals → at target form finer structures such as synapses, bouttons, etc. → connect source and target cells; a neuron can have many of these that move to find targets like other neurons or muscles

Complex Multicell Structures

self-assembled using basics of cell migration machinery coordinated with cues from the local microenvironment then need to be maintained

Actin and Tubulin (microtubules) filaments

both are polarized with specialized directed motor proteins; both bind porteins that control their stability; tubulin is anchored at the centriole and is a GTPase that slowly converts GTP to GDP

Permissive Factor

factors that are required for any movement; ex. f-actin; (for humans and cars - shoes, map/GPS, gas/lunch); “activate” a cell prior to its polarization and movement (ex. glucose); provide a polarity/prime the cell

Instructive Cue

cues that are required for directional movements but not movement itself (what is it from teh outside world that tells the thing where to go); street lights, signs, “path”; provides polarity within the cell that is already prepared for directed movements

Duty Cycle

steps: cell must polarize → directed polymerization of F-action at leading edge → attachament of leading edge to the substratum/substrate (extending like catching something with a rope or lasso) → contractivity as rear pulls cell forward and rear detaches → keep repeating for as long as the signal persists (if teh signal stops, the cell will get distracted and stop heading in that direction); if using a pipette to pipe a chemoattractant, that is an instructive cue

Experimental Tests

knockout, widespread over expression, and perturb or relocate gradient of signaling “cue”

Knockout

does not distingush whether the factor is permissive or instructive bu it does tell you if the protein (or whatever you are testing) is a factor or not; if motility is broken when the factor is removed, then it is a motility factor

Widespread over expression

elimnates the possible graident of an instruction; a lot of concentration of the factor (no gradient now for teh cell to follow)

Perturb or relocate gradient of signaling “cue”

changes the direction of cell movements; relocate gradient and see if cell will move with it (if it does → instructive cue); this is teh gold star test

Testing the Duty Cycle

proteins do not change they are just organized and activated in different ways; stationary - polarized all the way around but when it gets to matuartion it is only polarized at the leading edge

Different Forces Involved in Cell Migration

protrusion of membrane lamellipodia or filopodia requires force production (polymerization causes force); contractivity from detachament of the cell rear after protrusion becomes apparent to the substratum; traction at front can be low as long as contraction at the back is high

Integrin Adhesion Receptor Dynamics in Cell Migration

membrane and adhesion factors need to move as much as F-actin and myosin II; duty cycle

Proteins in Cell Migration

cell polarization (side/rear - PTEN and myosin II, front - activated Cdc42 and Rac, PIP3, activated integrin MTOC/golgi, and microtubules), protrusion and adhesion formation(nucleation - Arp2/3 complex, WAVE/WASP, and Rac/Cdc42, polymerization/organization - profilinm ENA/VASP, ADP/cofilin, capping proteinsm and crosslinkers), and rear retraction (adhesion disassembly and retraction - FAK/Src/ERk, myosin II, microtubules, Rho, Ca2+, calpain, and calcineurin)

Gradient and levels of G-proteins RAC activity control what

cell velocity and persistence

Optogenetic Control of RAC

LOV and J-alpha allow plants to control photosynthesis

LOV

light oxygen voltage sensing protein domain from plants

J alpha

inhibitory protein module