Final Flashcards
L20. Briefly describe the process of phagocytosis. What is the fate of materials engulfed by
phagocytosis
Phagocytosis can be considered to consist of 3 stages:
(1) Entrapment: the cell extends pseudopods (“false feet”) to contact and surround its
“prey;”
(2) Engulfment: the pseudopod membranes fuse, internalizing the prey in a “phagosome;” and
(3) Digestion: the phagosome fuses with a lysosome (or Golgi vesicles deliver lysosomal
enzymes to the phagosome) converting it to a “phagolysosome.”
In the phagolysosome, the “prey” is digested by hydrolytic enzymes (proteases, nucleases,
lipases, glycosidases), and the resulting products transported to the cytoplasm for recycling.
L20. What is autophagy?
Autophagy literally means “self eating.” In autophagy, cells surround worn
out organelles with specialized regions of ER, enclosing them into “autophagosomes.” From there,
they are delivered to lysosomes for recycling of their components.
L20. When starved for amino acids, cells of the Baker’s yeast Saccharomyces cerevisiae
upregulate autophagy, internalizing and digesting their own plasma membrane and plasma
membrane proteins. Why might this be beneficial to the yeast cells?
Autophagy of the plasma
membrane (and membrane proteins) and organelles provides a source of amino acids for the cell to
survive periods of starvation (albeit briefly)
L20. Diagram/compare the mechanisms of pinocytosis of lucifer yellow (LY) and receptormediated
endocytosis of low density lipoprotein (LDL). Label the important intermediates and
the compartment in which LDL is uncoupled from receptor (LDL-R), and the fate of the LDL-R.
Diagram
Both processes use clathrin-coated vesicles to internalized molecules from outside the cell.
The formation of clathrin coated vesicles in both processes is the same:
(1) Assembly of a coat of adaptins and clathrin, driving invagination and formation of a coated pit. In
“receptor-mediated” endocytosis, receptors are recruited to the coated pits via interactions with
adaptins;
(2) Formation of a clathrin-coated vesicle, requiring GTP hydrolysis by dynamin;
(3) Uncoating, requiring ATP hydrolysis by the “clathrin-uncoating ATPase” (an HSP70 family
member) ;
(4) Delivery to the early endosome. In pinocytosis, material enters the clathrin-coated pit/vesicle simply by diffusion. Thus the rate of internalization is highly dependent on the extracellular concentration of the molecule being taken up (at low concentration, very few molecules will end up in each vesicle). The presence of a cell surface receptor ( thus, “receptor-mediated” endocytosis) serves to concentrate low abundance molecules into coated pits/vesicles for more rapid/efficient uptake.
L20. What is the source of energy for uncoupling LDL from LDL-R?
Energy for uncoupling of LDL from receptor in the early endosome is provided by a pH
gradient (the endosome is acidic relative to the cytoplasm), created by an ATP-driven proton pump
in the endosomal membrane.
L20. What stage(s) of LDL uptake might be blocked by mutations that inactivate: (1) the
cytoplasmic domain of LDL-R? (2) the adaptin complex? (3) dynamin? Explain each.
- Mutations that inactivate the the cytoplasmic domain of LDL-R disrupt recruitment of LDL-R to
coated pits. - Mutations that inactivate the adaptin complex would block assembly of the clathrin coat required
for internalization. - Mutations that inactivate dynamin would block the final budding of the vesicle
L20. Non-hydrolyzable analogs of GTP, such as GTPS or GMPPCP, inhibit the formation and/or
function of both COP-coated and clathrin-coated vesicles. Compare the role of nucleotide
hydrolysis in the assembly/disassembly of COP- and Clathrin-coated vesicles, indicating which
nucleotide provides the energy for assembly/disassembly of the vesicle coat, how hydrolysis is coupled to the cycle of assembly/disassembly, and what cellular protein/enzyme functions as the
“NTPase.”
COP-coated vesicles:
A GEF in the donor membrane activates the coat recruitment GTPase (either and
ARF or Sar1) by facilitating exchange of GTP for GDP.
The active, GTP-bound form of the coat recruitment GTPase binds to the donor
membrane and recruits COPs, which cause vesicle budding.
Hydrolysis of GTP by the coat recruitment GTPase releases the GTPase and COPs,
uncoating the vesicle. GTPS blocks UNCOATING.
Clathrin-coated vesicles:
Adaptins and clathrins assemble on the membrane to form clathrin-coated pits,
recruiting cargo receptors and their ligands…
GTP hydrolysis by the GTPase DYNAMIN is required for the final budding/scission
deeply-invaginated coated pits to form the coated vesicle. So GTPS blocks vesicle
SCISSION/FORMATION, but NOT coating or uncoating.
ATP hydrolysis by the CLATHRIN-UNCOATING ATPase is used to remove the
clathrin coats within seconds of vesicle formation.
L21. Because of the cell-type specificity of intermediate filament subunit proteins (IFPs),
antibodies against IFPs can be used to help identify the tissue of origin of human tumors. This
can greatly improve the accuracy of diagnosis and treatment, often improving a patients
prognosis. What type of intermediate filament protein might you expect to find in:
A. a neuroblastoma (tumor of neuronal precursor cells)?
B. a basal cell carcinoma of the epidermal epithelium?
C. a rhabdomysosarcoma (muscle)?
D. a glioma (tumor of glial cells)?
A.Neurofilament Proteins
B. Keratins
C. Desmin
D. Glial Fibrillary acidic protein (GFAP)
L21. Unlike actin filaments and microtubules, intermediate filaments are not “polar.” What does
this mean with regard to intermediate filament structure and function?
IF proteins assemble in parallel to form dimers, but dimers associate in an ant-parallel
orientation to form tetramers. In the filament cross-section, there are 8 tetramers. Because there
is an anti-parallel intermediate AND an even number of subunits in the unit filament, the two ends
of the filament look the same (thus the filament is not “polar.”). This contrasts with actin filaments,
where the actin monomers assemble head to tail, giving the filament two ends with distinct
properties (“plus- (barbed)” and “minus- (pointed)” ends), and microtubules composed of 13
protofilaments arranged in parallel.
There are no motor proteins that use IFs for a substrate to generate force… this MAY
(stress may) relate to their lack of directionality… or it may have other more mundane evolutionary
reasons.
L21. Sketch the organization of a skeletal muscle myofibril, including thin and thick filaments,
the Z- and M-lines. Indicate the polarity of the thin filaments. What are some of the major
protein components of each structure?
DIAGRAM
L21. In 10 words or less, what is the basic mechanism of muscle contraction?
ATP-dependent sliding of actin and myosin filaments.
L21. Myosin and HSP70 are very different proteins with very different functions in the cell.
What common functional characteristic do they share?
Both proteins are using nucleotide hydrolysis to power a cycle of making and breaking highaffinity
protein-protein interactions. HSP70, by binding and releasing hydrophobic regions of
unfoled/misfolded proteins & myosin by binding and releasing actin
L21. Briefly outline/discuss the steps of the “crossbridge cycle” by which myosin uses the energy of ATP to “walk” along the actin filaments, generating muscle contraction.
Diagram
- No nucleotide. Myosin head is tightly bound
to actin (“rigor”)… - ATP bound to myosin releases myosin from
actin… - ATP hydrolysis “cocks” myosin (ADP + Pi
remain bound). Myosin binds actin weakly… - Pi is released, allowing myosin to bind more
tightly, triggering… - …the “power stroke” and release of ADP…
Myosin heads “walk” towards the “plus-”
(“barbed”) -end of actin filaments…
“Myosin is an actin-dependent ATPase that acts as a “molecular motor””
L21. The post-mortem condition know as “rigor mortis” is characterized by stiffness and rigidity of all the skeletal muscles. Based upon your understanding of the molecular interactions of myosin and actin, propose a molecular explanation for rigor mortis.
Once ATP is depleted, all myosin heads will bind tightly to actin, locking all the muscles into
a rigid state known as “rigor mortis.” At the molecular level, cell biologists refer to the bound state
in the absence of ATP as “rigor.”
L21. Why do you think rigor mortis subsides several hours after death?
As muscles and other cells die, organelle membranes lyse. Proteases released from the
lysosomes cleave myosin and actin, “tenderizing” the muscle (this is why “aged” beef is more tender).
L21. How does Ca2+
regulate contraction
of skeletal muscle in
vertebrates?
Diagram
Tropomyosin dimer binds along actin filament…
Troponin complex binds to tropomyosin…
In the absence of Ca2+, tropomyosin blocks
myosin binding site…
Troponin C binds Ca2+…
Allosteric change in structure of troponins and
tropomyosin uncovers myosin binding site…
Myosin walks on actin and myofibril contracts…
Removal of Ca2+ restores inhibition…
L21. Where does the
Ca2+ come from?
Ca2+ is stored in the “sarcoplasmic reticulum,” and released through voltage-gated Ca2+ channels in response to an action potential
L21. In the common “sliding filament” assay for myosin
motility, myosin molecules are adsorbed to a glass
coverslip. Fluorescent actin filaments are bound to the
myosin-coated coverslips. When ATP is added, the actin
filaments glide along, with a defined polarity and direction
of motion. If a single headed type I (unconventional)
myosin was adsorbed to the coverslip, which end of the gliding actin filaments (“pointed /minus”
or “barbed/plus”) would lead?
Myosins walk towards the plus-end of actin filaments, so the minus- end would lead
L21. In the large internodal cells of some freshwater
algae, an inner layer of cytoplasm “streams” around the
central vacuole at rates >1 m min-1. Streaming is
powered by myosin-dependent sliding of
vesicles/organelles along actin filaments at the interface
between the stationary cortical cytoplasm and the
streaming layer (see fig inset). If the polarity of the actin
is as shown in the figure (plus “+” or minus “-“) , which
direction would the vesicle in the figure move (Circle
the arrow, and briefly explain in <10 words; 2 pts).
Diagram
Myosin walks towards the plus end of actin filaments
L21. Sketch the characteristic organization of actin microfilaments in the lamellipodium of a
migrating animal cell, indicating/identifying (A) the polarity of at least two actin filaments; (B)
an ARP 2/3 complex; (C) where actin monomers are added to those filaments; and (D) the site of
actin disassembly. (E) Briefly describe the role of actin and ARP2/3 in lamellipodial extension
and cell motility
Arp 2/3 is activated near the inner face of the plasma membrane (by Rho family GTPases and other activating proteins)... Activated Arp 2/3 complex nucleates actin assembly and branching. Arp 2/3 caps minus-ends. Assembly of the extensively branched meshwork of actin filaments drives extension of the lamellipodium. Actin filaments then disassemble (from their minus-ends) behind the lamellipodium...
L21. What is the difference between an actin cross-linking protein and an actin bundling
protein? What are examples of each?
Most actin bundling proteins are rigid rod-like molecules with an actin binding
domain on each end, allowing them to organize actin filaments into parallel bundles.
A good example is a-actinin.
Most cross-linking proteins are longer, more flexible proteins. Again, they have
multiple actin binding domains, allowing them to cross-link actin filaments. However,
since they are long and flexible, the angle of the actin filaments is more variable.
Good examples are filamin and spectrin
L21. Cells of the cellular slime mold Dictyostelium discodium express multiple actin bundling
proteins including -actinin and ABP. Interestingly, genetic disruption of either protein alone
has no observable effect, but disruption of BOTH proteins in the same cells resulted in marked
defects in motility and development. What would you conclude about the function of actin
bundling proteins in Dictyostelium?
The proteins may be REDUNDANT… meaning they perform
similar and overlapping functions. If one is defective, the other can take over its function.
L21. Briefly compare/contrast three different ways in which the actin cytoskeleton can be linked
to the plasma membrane of the cell. Which of these can be used to integrate the actin
cytoskeleton with the extracellular matrix?
(1) via integrins at focal contacts and adherens junctions: plus-ends of actin filaments linked to
integrins (transmembrane ECM receptor) via linker proteins…
(2) via spectrin complexes of the membrane cytoskeleton: junctional complexes in which actin and
spectrin are linked to glycophorin via band 4.1…
(3) via unconventional myosins, as 100 kDa myosin in brush border microvilli…
(4; not mentioned in lecture) to transmembrane proteins via ERM family linkers (not discussed in
lecture).
The actin cytoskeleton is commonly linked to the ECM via integrin family receptors at focal contacts
and adherens junctions
L21. Briefly sketch/compare/contrast the structure
and function of prokaryotic and eukaryotic flagella,
including and labeling the major structural
components of each briefly describing their function
in the generation of motility (what is the energy
source for motility, and what proteins/structures are
the motors?).
Diagram
Eukaryotic cilia and flagella are composed of a 9+2 arrangement of 9 outer doublet
MTs surrounding a central pair of MTs, located within the plasma membrane.
Motility is generated by ATP-dependent sliding of the outer doublet MTs powered by the “dynein”
arms. This sliding is converted to bending by nexin linking the adjacent outer doublets, and is
controlled by accessory proteins/structures of the flagella (central pair and radial spokes)
L21. How do eukaryotic cilia differ from eukaryotic flagellae?
Eukaryotic cilia are generally shorter
than flagellae, and have an asymmetric strokes. Flagella (euk) are often longer and have symmetric
strokes.
L21. What structural/morphological defects are apparent in pfA mutants? Briefly describe the
normal mechanisms for generating flagellar motility in eukaryotes, and explain why pfA mutants
have paralyzed flagellae
In normal flagellae, the dynein arms attached to A subfibers use ATP hydrolysis to power a
crossbridge cycle similar to that of myosin, walking towards the minus-end of the adjacent Bsubfiber
of the adjacent outer doublet. This MT sliding motion is converted to bending by the nexin
links between outer doublets.
Mutant pfA is missing its dynein arms. In their absence, the flagellae of pfA are paralyzed
L21. Figures C and D depict flagellar cross-sections from two other mutant classes (pfB and pfC)
isolated in your screen. What structural/morphological defects are apparent in flagellae from pfB
and pfC mutants? Briefly relate these defects to the mechanisms of flagellar motility
pfB is missing the central pair/sheath. pfC is missing the radial spokes. Sliding/bending is
temporally and spatially controlled by the central apparatus, including the central pair, sheath,
radial spokes, etc. In their absence, the flagellae are paralyzed.
L21. What structures contributed by the sperm during fertilization might function as centrioles to
organize the centrosome that nucleates the sperm aster?
The basal bodies of the flagella (which are structurally identical to centrioles, and recruit other
centrosome components from the egg cytoplasm).
L21. Microtubules are highly dynamic polymers, undergoing frequent interconversions
between phases of growing and shrinking. What is this behavior called?
“Dynamic instability
L21. What effect would you expect GTPS, a non-hydrolyzable analog of GTP, to have on the
dynamics of microtubule assembly in vitro? Explain your answer.
Current models of dynamic instability suggest “catastrophe” (the transition from growing to
shrinking) is caused by loss of the GTP-tubulin cap at the MT end. GTPS and other nonhydrolyzable
analogs of GTP stabilize MTs by slowing or preventing GTP hydrolysis and loss of the
GTP-tubulin “cap,” thus keeping the MT in a growing state.
L21. What class of proteins regulate microtubule dynamics?
Microtubule-associated proteins, or “MAPs
L21. If a kinesin-like motor was assayed in such a manner, which end of the MT would lead (plusend
or minus-end)?
Kinesin walks towards the plus-end, so the minus-ends would lead in this assay.
L21. If dynein was adsorbed to the coverslip and ATP was added, which end of the MT would lead
(plus-end or minus-end)?
Cytoplasmic dynein walks towards the minus-ends of MTs, so the plusends
would lead in this assay
L21. Based on your understanding of the cytoplasmic distribution/localization of the Golgi and ER
relative to MT organization, which motor do you think would power: Vesicle transport from the
ER to the Golgi? Vesicle transport from the Golgi to the plasma membrane?
Draw yourself a cell
with a perinculear Golgi and ER dispersed throughout the cytoplasm. Where is the centrosome?
What motor would be used for peripheral ER to Golgi (dynein… why?)? from Golgi to plasma
membrane (kinesin…why?)?
