Actin polymerized from actin protein (binds ATP)
(first found in muscle cells, but most kinds of cells have it)
Microtubules polymerized from tubulin (binds GTP)
(hollow, rather stiff)
(found inside cilia & flagella; but are in cytoplasm too)
Intermediate filaments polymerized from keratin, etc. etc.
(really include several different kinds of proteins,
that don't actually have that much to do with each other)
II) actin fibers and microtubules exert forces and create patterns
in two different kinds of ways:
A) active sliding B) polymerization (assembling into fibers)
A) because certain other proteins ("motor proteins")
slide along them:
(myosin slides actively along actin fibers)
(dynein slides actively along microtubules)
(kinesin (also) slides actively along microtubules)
This active sliding uses the energy from ATP
For example, in muscle, myosin slides along actin
and in cilia & flagella, dynein slides along microtubules
And there are many other examples of each:
for example, amoeboid locomotion mostly due to actomyosin
another example, mitotic spindles are made of microtubules
and dividing cells pinch in two using belts of actomyosin
B) By assembling monomers onto the ends of actin fibers and microtubules. NOTE how important it is that they add and lose monomers only at the ends of fibers
III) Procaryotes & arhcaea don't have actin or tubulin
but they do have a tubulin homolog called FtsZ
(pronounced Futz-Zee) filamentous mutant # Z
A thought question; if bacteria can't divide, they form filaments
but why should mutation of this gene cause filament formation?
IV) Actin protein binds ATP (or ADP) tubulin binds GTP (or GDP)
Being bound to the tri-phosphate promotes assembly into fibers
Being bound to the di-phosphate promotes DISassembly,
from fibers back into monomers
Imagine one of these proteins diffusing around the cytoplasm, binding to an ATP, then assembling onto the end of a fiber
(but while in the fiber, cleaving the ATP to ADP; thus tending
to disassemble back out of the fiber again
V) Microtubules and actin fibers ends differ
Each microtubule has a plus end and a minus end
Each actin fiber has a plus end and a minus end
THIS HAS NOTHING TO DO WITH + OR - CHARGES
Plus ends where addition (and also removal) of subunits favored
Minus ends are where assembly & disassembly are slower.
{Large numbers of people, including some textbooks, think that disassembly is favored at the minus end; almost might as well be!}
VI) Two related kinds of dynamic behavior result from this:
A) Treadmilling (on one end (+), off other end (-))
B) Dynamic instability (irregularly on & off same end, mostly+)
microtubules frequently have dynamic instability
actin fibers frequently treadmill (in cell locomotion, for example)
(in principle, however, either could do either) (I think!?)
In the time lapse video that you saw showing microtubules
they were elongating and shortening by dynamic instability
(at their + ends)
And they were being pushed rearward by a constant wind of treadmilling actin fibers (+ end at cell edge, - end in center)
VII) There are lots of special proteins whose function is to bundle actin fibers together; other special proteins to cut them;
other special proteins to nucleate their formation,
or to stabilize their ends, in the sense of blocking disassembly
and likewise for microtubules
(& that is how cells control their shapes & movements, etc.)
VIII) There are special poisons that bind tubulin & actin
These are useful for experimenters, & also used to treat cancer.
vinblastine, taxol, nocodazole & colchicine
are some of the main tubulin poisons
phalloidin and cytochalasin are actin poisons,
(phalloidin is from deadly nightshade mushrooms, & they also have RNA polymerase poisons)
IX) Myosin is a + end directed motor protein (along actin)
(Besides the 2-headed kind of myosin found in muscles
are other myosins that transport stuff around the cytoplasm)
UNC Prof. Dick Cheney {no relation to the V. Pres) is the world's best researcher on these "unconventional myosins"
Kinesin is a + end directed motor protein along microtubules
Dynein is a - end directed motor protein along microtubules
..... all 3 of these proteins are ATPases
Dynein drives bending of cilia and flagella:
& also drives transport of membranes, organelles etc.
toward the middle of cells (- ends of microtubules)
Kinesins drive transport of organelles toward cell edges
(where the + ends of the microtubules are...
especially important in carrying stuff to ends of nerve fibers.
X) Most animal cells can crawl: especially in tissue culture,
during embryonic development, in cancer invasion
The reaching, sticking, contracting sequence (fig 16-85) is wrong
really, they use actin treadmilling to pull material rearward
past their upper & lower plasma membranes
Traction forces: which can distort rubber & carry particles
fig 16-94 & 16-91
& can also reorganize extracellular collagen to make tendons
fig 16-95 (which by some accident is the same as fig 19-50! rotated 90 degrees!)
XI) Certain GTP-binding proteins: Rho, Rac & Cdc42
serve to control actin fiber organization
and are very important in cancer research. ("Rac and Rho are here to stay")
Programmed cell death = "apoptosis"
#1) Is NOT the same thing as ordinary cell death ="necrosis"
instead, apoptosis is a deliberate self-destruction, in which
special enzymes digest the cell from the inside out.
The classic example of programmed cell death is the destruction of the tail of tadpoles, as they change themselves into frogs
#2) The main mechanism is activation of special proto-enzymes
in the cytoplasm: called caspases
(because they have aspartic acid and cysteine at their active sites)
The active site is blocked by part of the protein chain;
but once this part is digested away, then caspases become active
activated caspases can unblock other caspases: a domino effect
{please note: in case you are ever looking for a cure of cancer,
all our cells (including all cancer cells!) already have this self-destruct system ready and waiting inside them, if you can set it off!}
#3) Other examples of apoptosis include
A* elimination of ~1/2 of embryonic nerve cells.
B* elimination of webbing between vertebrate toes.
C* getting rid of many specific cells in nematode embryos
D* weeding out lymphocytes that make anti-self antibodies
E* killing virus-infected cells (even in colds, etc.)
F* much of the cell death after strokes & heart attacks!!!
(& also nerve cell death in severed spinal cords!)
Therefore, it might be a big medical advance if somebody were
to discover a pill etc. to prevent all apoptosis for a few days;
that might prevent up to 90% of the long-term damage.
The same basic mechanism of apoptosis is used in all these cases
(but plants have a different system to protect from virus infection)
#4) There are some special cell proteins whose function is to initiate apoptosis: Fas and Fas ligand (produced by killer lymphocytes)
#5) A special set of (mitochondrial !) proteins serve to inhibit apoptosis Bcl-2 and the Bcl-2 family (Bad, Bax etc.)
These were discovered in human B-cell lymphoma cancer patients,
because chromosome breaks that put the Bcl-2 gene next to active promoters cause too much of this protein to be made,
& such lymphocytes accumulate & can't die; causing lymphoma
(but such cases of lymphoma are still treated with anti-DNA growth poisons; which logically should be no help! But do cure some!?
Several viruses make their own Bcl-2 like proteins, to help protect themselves from self destruction of infected host cells.
A certain gene discovered in nematode worms resembles bcl2
so much that substitution of the human gene into worms results in a normal phenotype. Nobody tried the reverse experiment!!
The cell cycle: the Nobel Prize for last fall was for cyclins, etc.
#6) DNA synthesis is done during a specific period between the times of actual cell division (mitosis). (in eucaryotes!)
This was an early discovery of radioactive precursors
M period
G1 period
S period this is called "the cell cycle"
G2 period
M period
#7) Progress from one phase to the next is controlled by several kinds of "checkpoint mechanisms" (often important to cancer etc.)
There are mitotic checkpoints - that halt mitosis if chromosomes aren't lined up
There are DNA damage checkpoints that halt DNA synthesis until damage is fixed
If damage can't be fixed, checkpoint mechanisms set off apoptosis
#8) Timing of each phases of the cycle turns out to be
controlled by synthesis & accumulation of specific proteins.
these proteins are named cyclins
(there are different cyclins for the G1 period, etc.)
this is sort of analogous to an hour-glass, except the cyclins are made & destroyed
When the concentration of a cyclin becomes high enough, this sets off two events:
one-> going on to the next stage
two-> digestion of all of that accumulated cyclin protein
These events are set off by cyclin-dependent kinases (enzymes)
(tell the story about how cyclins were accidentally discovered by a lab project in the Woods Hole physiology course: short term labeling)
#9) When cells halt growth, it is (almost?) always at the boundaries
between phases of the cell cycle; usually at the G1-S boundary
G-0 pronounced Gee-Zero
Certain proteins called growth factors, and other "mitogens"
can push cells through this boundary.
including "platelet derived growth factor" (derived from blood platelets)
The internal mechanisms of this G1-S checkpoint
include RB protein, and others that check for DNA damage
#10) frogs & flies (but NOT humans or other mammals)
turn off their checkpoint controls during early cell cycles
(so their embryos have no G1 or G2
they just alternate between M and S, until ~ thousand cells.
Some unsolved problems: related to sizes of individual cells
Tetraploid cells become exactly twice the volume of diploid cells;
and haploid cells become exactly half the size of diploids.
Species with lots of junk DNA have proportionately bigger cells!
Organ sizes and shapes seem to be independent of cell size.
fig 17-52
#II) Animals, plants & all other eucaryotes form a special structure
The mitotic spindle (made mostly out of microtubules)
microtubules radiate out from 2 asters (MTOCs) centrioles
and their chromosomes condense
(DNA strands coil up and become visible
(and DNA becomes inactive, transcriptionally)
Mitosis is divided into the following stages, that you should learn..
prophase (chromosomes condense, spindle forms)
metaphase (chromosome pairs, kinetochores line up along midline
(where they line up is called "metaphase plate")
anaphase (chromosome pairs pulled apart, toward opposite poles)
telophase (chromosomes "decondense", new nuclei form, etc.)
There are some eucaryotes with interesting variations on this, such as that the nuclear membranes sometimes remain intact, etc.
Dinoflagellates, diatoms, yeasts and other interesting variations. 18-41
#III) Animal cells split in two by contraction of a contractile ring of actin and myosin that forms just under the plasma membrane, around the "equator" usually around where the metaphase plate was.
BUT IN CONTRAST
Plant cells separate by building a new cell wall (exocytosis) by a
"phragmoplast" that forms (usually) where the metaphase plate was
the cell plate, etc. but the future location of the phragmoplast
is where the preprophase band of microtubules had formed in prophase?
So: no contractile ring in plants; no phragmoplasts in animal cells
& neither one in bacteria; and no mitosis or spindle in bacteria.
#IV) There is a lot of research about what signals control where the contractile ring forms. "cleavage furrow"
A) Signals come from the poles (not the equator)
Ray Rappaport's classic "doughnut" experiment fig 18-31
B) The contractile ring is a concentration of actin and myosin
C) 100s of papers have been published debating subjects as whether
the signals from the poles cause stronger or weaker contraction...
Julie Canman in this dept, THIS WEEK, may have discovered the answer to several of these questions, about how asters control the location where the contractile ring!! (in PTK1 cell line:rat kangaroo)
She used a certain kinase inhibitor to cause cells to form
monopolar spindles (only one aster, instead of 2)
and she injected fluorescent tubulin, and made video time lapse
to record the cells' behavior: The new discovery is that cleavage furrows formed and pinched off the ends of cells, further away than the lengths of aster microtubules.
This implies that something released from or at the ends of aster microtubules is what causes acto-myosin to aggregate into the ring.
#V) Anaphase A is pulling of chromosomes toward the poles
Anaphase B is elongation of the poles = spindle elongation
the relative degree of A vs. B varies widely between species
#VI) Poleward flux of microtubules, from kinetochore toward pole
during metaphase (look at fig # 18-21)
#VII) balance of forces mechanism (Ostergren, Hays, Skibbens)
causes chromosomes to gravitate to metaphase plate.
#VIII) Spindle attachment checkpoint delays anaphase until all kinetochore pairs are stretched at metaphase plate.
#IX) Two main hypotheses about the mechanical force that pulls the kinetochores toward the poles in anaphase:
* some kinesin-like motor, using ATP, in the kinetochore
* simply the depolymerization of the microtubules
#X) in early embryos of some species (flies, etc.)
mitoses occur without cytokinesis fig 18-36
13 rounds of cell cycles: mitoses produce< 6000 nuclei
then simultaneous mass-cytokinesis: hexagonal network of acto-myosin
1) Cell walls are made of secreted polysaccharides (chains of sugars)
(& are outside the plasma membrane)
2) Turgor pressure: osmotic pressure, of water "trying" to diffuse into cells, pushes plasma membranes outward against the cell walls.
This force is often very large: 22 atmospheres (700 foot depth in water)
burst bacteria, penicillin blocks enzymes that synthesize cell walls
3) In higher (multicellular) plants, the mitotic divisions are concentrated (only) in certain small zones = meristems
shoot meristems, root meristems, cambium (sideways meristem)
but divisions exert negligible (if any) pushing forces.
Plant shape is caused by controlled osmotic swelling;
by controlled weakening of cells walls: certain places & directions
4) Cellulose: polymer of glucose:
enzyme that synthesizes it is actually in the plasma membrane
and (apparently is guided by a layer of cortical microtubules
Cellulose fibers are often lined up all in one direction;
microtubules just under plasma membrane aligned in same direction
Later; MTs (somehow) become reoriented perpendicular to this;
and then the cellulose fibers are laid down parallel to this new direction
?What would you expect to happen in plant cells treated with colchicine?
Plants also secrete many other structural polysaccharides:
Lignin, pectins swelling: jelly
Cells in higher plants are connected by narrow cytoplasmic connections
called "plasmadesmata"
A thought question: what do you suppose was the evidence behind our textbook's statement that at least 700 of Arabidopsis' genes serve to control some aspect of cell wall morphogenesis?
5) Animal cells (no cell walls!!!)
extracellular matrix collagen (jello; glue; Elmer's cement
Glycine every 3rd amino acid almost 1/3 proline
triple helix (NOT alpha helix pattern) long, stiff, side links
Type I collagen, type II collagen, type IV collagen, into the 20s
67 nm repeat striations
tendons, dermis, organ capsules, walls of arteries, tooth-jaw connections
often form alternating perpendicular layers: cornea, intervertebral discs
vitamin C needed for secretion of collagen scurvy
collagenous structures therefore can't be renewed
the ones with the fastest turnover degenerate first.
6) Integrin (transmembrane connection) from actin to fibronectin
integrin a dimer of alpha & beta components, many interesting facts
fibronectin is an extracellular protein: connects collagen to integrin
RGD peptide snake venom "disintegrin"
(there are lots of other interesting proteins in snake venoms)
Focal adhesions: focal adhesion kinases, etc.
7) mechanical interactions between fibroblast cells and collagen
note figure # 19-50; which is also fig # 16-95 (rotated 90 degrees)
The theory that fibroblast traction is the mechanism that causes
formation of tendons, capsules, wraps blood vessels, etc.
But what could be the mechanism that forms the 90 degree/layers?
8) other matrix components:
9) Epithelia basement lamella laminin type IV collagen
differences between apical versus baso-lateral plasma membranes
10) Different kinds of cell-cell adhesions (especially in epithelia)
tight junctions (occluding junctions: also control membrane proteins)
Gap junctions connexin proteins
(mention electrotonic coupling of heart muscle cells to each other)
Desmosomes
adherens junctions cadherins cell adhesion molecules
selective adhesion: cadherin E, cadherin N, cadherin P etc.
and several other selective adhesion proteins
Cell sorting ; H.V. Wilson, and all that: fig 19-27