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Summary of Cell Organization & Motility (Lecture 20 / 24M Feb, 2014)

1) Cell organization
Cells come in many different shapes, but ALL have the same genome! How?
o Skin cells -> Stratified squamous tissue
o Cheek cells -> Flat shaped
o Muscle cells -> Long, multinucleated & tubular
o Neurons -> Vary greatly
Cells are internally organized (eg. diff. apical & basolateral domains in epithelial cells).
o Cells achieve this via polarized distribution of proteins, which allows internal
organization (rather than maintenance of structures in a
uniform mixture) and ensures localization of specialized
structures, such as microvilli, on the apical domain.
Cells import & export cargo via motor proteins along microtubules

2) Motility of cells
Cells can transport themselves thanks to their internal skeleton, aka cytoskeleton.
o Wound repair: Certain cells (e.g. neutrophils) migrate from blood vessels to the wound to help
with tissue repair or immune response.
o Tumor metastasis: cells walk out of the primary tumor,
break through the blood vessel wall and establish
themselves in new tissues.

3) Cell cytoskeleton
Cells have proteins that can reorganize their skeleton and thereby move the cell through space.
The cytoskeleton plays a key role in cell physiology & metabolism. It is responsible for:
o Shape, structure, stability
Breaking & re-building cytoskeleton molds shape of organism/organelle.
o Intracellular transport & (Internal) spatial organization
o Contraction & motility of the cell (incl. muscle contraction)
Made up of 3 major polymer systems that have the ability to self-assemble:


Microfilaments
(aka actin filaments)
Microtubules Intermediate filaments
Subunit Actin -tubulin dimer Various
Structure

(filament-shaped)


(rope-like polymers)
Location
a) Underneath cell
membrane
b) Spreading across entire
cell (internally)
Near the nucleus
Very heterogeneous polymer
group -> various locations
e.g. lamins help form the
nuclear lamina/basket
Study
Interest
Most studied Average
Least studied, even if their
breakdown causes many
laminopathies (eg. skin diseases)
Properties
Similar to wires; very high
tensile strength (can bear a
lot of load when pulled) &
flexible (low flexional
rigidity).
Similar to poles in
terms of mechanics;
very stiff, dont like to
bend (high flexional
rigidity) & resistant to
compression.
Similar to ropes; elastic (stretch
when pulled on & return to
original shape when released),
very flexible (bent very
easily).

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Cytoskeleton is a tensegrity structure built on tensional integrity; it maintains shape by pulling
in many directions, rather than via gravitational compression how buildings maintain shape.

4) Actin cytoskeleton
Actin is an ATPase with the ability to self-assemble, and by
doing so it drives cell motility.
Each actin monomer = 4 lobes & 1 ATP-binding cleft.
Actin polymerizes into a 2-stranded helical filament with a
periodicity of 36nm. Having multiple strands promotes
stability.
All actin monomers & the full polymer are asymmetrical.
o Each monomer has 1 end with a cleft and 1 smooth end. The smooth end
faces the strands (+) end, while the cleft end faces the (-) end.
Actin (with myosin) is a major component of muscle (see figure).
Actin filaments are arranged differently at diff. times & intracellular locations.
o Microvilli: projections from intestinal epithelial cells made of actin bundles.
o Cell cortex: network of actin directly underneath the plasma membrane.
which provides mechanical support to plasma membrane (found in all cells).
o Adherens belt: cortical band organized into a belt where cell-cell
adhesions occur (in some cells).
o During migration, actin helps form protrusions called filopodia,
lamellipodium/leading edge* & stress fibers.
*Branched, dendritic network of actin filaments
o Actin forms cross-linked structures during phagocytosis and
near moving endocytic vesicles.
o Also forms a contractile ring during cytokinesis.
Cells are able to control actin on diff. levels: length, number, angle
(lining up or crossing at skew angles), bundling (e.g. parallel actin
bundles in microvilli & stress fibers) & orientation (e.g. many short,
branched dendritic network of actin filaments form the leading edge).

5) Actin filaments driving cell movement
Cell moves forward via outward polymerization of actin, while stress
fibers pull up the back end.
Focal adhesions are protein complexes that allow the cell to attach to
surfaces and acts as the cells feet.
Step 1) Actin filament polymerization allows formation of lamellipodium &
extension of plasma membrane forward in direction of movement.
Step 2) Formation of a new adhesion.
Step 3) Translocation of cell body (shifting forward).
Step 4) De-adhesion of the lagging foot and endocytic recycling of those
components in order to allow future formation of new adhesions

6) Actin polymerization
Occurs in 3 phases:
o 1) Nucleation: 3 free G-actin (globular
actin) come together to form a nucleus.
This is a difficult phase since 3 subunits need to fuse in the right orientation almost
simultaneously (dimer would fall apart).
o 2) Elongation: Actin monomers, now called filamentous F-actin, attach themselves to form
long filaments (faster than nucleation / 1 monomer at a time).
o 3) Steady state: nb of actin subunits joining the polymer = nb of actin subunits coming off.
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Equality is reached b/c the nb of free G-actin drop gradually with polymerization.
Mass of the filaments plotted as a function of time:
o The slope initially increases slowly due to the difficulty of nucleation. Elongation then
rapidly increases the mass until the steady state is reached.
Actin polymerization is described by rate constants.
At the (+) end of the actin filament:
o On-rate constant:
Rate: 12 M
-1
s
-1
(e.g. for a solution with 1M of
actin, 12 actin subunits would attach per sec)
Concentration dependent
o Off-rate constant:
Rate: 1.4 s
-1

Concentration independent due to the
collision b/w other agents in the solution
& the terminal actin subunit
o Critical concentration (C
+
c) at which on-rate = off-
rate: 0.12 M (since 0.12M x 12M
-1
s
-1
= 1.4s
-1
).
At the (-) end of the actin filament:
o On-rate constant:
Rate: 1.3 M
-1
s
-1

10x lower than at the (+) end
o Off-rate constant:
Rate: 0.8 s
-1

o Critical concentration (C
-
c): 0.60 M
Overall, the net growth of the actin filament can be described as such:
o Net Growth = Subunits added Subunits lost = k
ON
[subunits] k
OFF (1).

The critical concentration is: C
c
= [subunits] when Net Growth is zero
o From (1), C
c
= k
OFF
/ k
ON
Critical concentrations at either end are different:
o At C
-
c, the (+) end will not be satisfied and will gain more subunits than it loses. As the
concentration of free subunits drops and C
+
c becomes satisfied the (-) end starts to lose
more subunits than it gains.
o Real steady state: Growth at (+) end = depolimerization at the () end (2).
This happens at a concentration of actin monomers in b/w C
+
c and C
-
c (0.165M)
This value is found by adding association rates (13.3 M
-1
s
-1
) & dissociation
rates (2.2s
-1
), then substituting these into (1), giving 2.2/13.3 = 0.165M OR
Isolate [substr.] from equation (2): 12*[substrate]-1.4 = -(1.3*[substrate]-0.8).
Actin polymerization includes an intrinsic ATPase activity. Actins ATPase cycle -
o Step 1) When an actin monomer comes on, it has an ATP in its ATP binding pocket. Thus,
the (+) end is enriched in ATP-actin.
o Step 2) After the actin monomer comes on, its ATP gets hydrolyzed, which gives a 2nd
region of filament, comprised of ADP-Pi-actin.
The phosphorylated bond was broken, but Pi has not been released yet.
o Step 3) ADP- Pi-actin releases its Pi later while still part of the filament, marking the start of
the final segment of the filament, comprised of ADP-actin.
Actin Treadmilling at a steady state
o Length of actin filament remains the same, but filament translocates forward b/c (+)end
grows at steady state while (-)end shrinks. This is known as actin treadmilling.
Thus, cells control actin polymerization to drive motility.
Possible questions to consider: Speed of treadmilling? Concentration of monomers at steady-state?