Lecture 2 - Quiz 1 Flashcards
What is the cell interior filled with?
Organelles, cytosol and cytoskeletal components
How can you visualize the cytoskeleton?
Treat cell with detergent like Triton X to extract soluble proteins of cytosol and fix and observe with SEM
What is the cytoskeleton?
A network of protein filaments throughout the cytosol that goes around or between organelles and can help with structure and transport
What has the greatest surface area within a cell?
Cytoskeletal network is >12x the SA of the combined membranous components of cells
What filaments make up the cytoskeleton?
Actin, intermediate and microtubules
Rank the cytoskeleton filaments by size
Actin (7-9 nm), intermediate (10 nm), microtubules (24 nm)
What is another name for actin filaments?
Microfilaments
Which of the filaments is not found in bacteria or fungi?
Intermediate filaments
How can filaments be organized structurally?
Bundles (parallel), geodesic-domes (found under membranes with foci radiating out), or gel-like 3-dimensional lattices
What are some functions of the cytoskeleton?
Organize the cytosol and establish polarity, support specialized structures, support the plasma and nuclear membrane, cell motility
What is an example of specialized supported structures by the cytoskeleton?
Microvilli, cilia, flagella
How does the cytoskeleton support the plasma and nuclear membrane?
Microfilaments indirectly attach to the plasma membrane with accessory proteins by forming geodesic dome structures
How does cytoskeleton provide indirect linkages between adjacent cells and cells and the ECM?
Actin and IFs provide adhesion by associating with adhering junctions and demosomal junctions by hooking in to junctions
How does the cytoskeleton provide intracellular movements?
Microtubules can move vesicles, organelles and chromosomes, actin can also participate
How does the cytoskeleton provide cell motility?
Microtubules form core of cilia and provide structural support for ciliary beating and actin can utilize motor protein myosin to generate force for motility, flagellar beating of sperm
Which is the most abundant intracellular protein?
Actin
What are the forms of actin and what property does this give actin?
Monomeric, globular G-actin and polymeric, filamentous F-actin, allows actin cytoskeleton to be dynamic and change shape and size
What is the structure of G-actin?
It is non-symmetrical with 2 lobes with a cleft between where ATP can bind and be complexed with magnesium
What is the structure of F-actin?
Binds in tight helix of twisted strand of beads and strand has polarity with units always binding with ATP binding cleft facing negative end
How can you distinguish the negative and positive end of F-actin?
Myosin decoration forms arrowheads
How does actin polymerize? What are the steps?
G-actin aggregates into short, unstable oligomers during nucleation or the lag period
Stable oligomers 3-4 subunits long stabilize during elongation and free g-actin decreases
Steady state is achieved when there is no net increase in monomers and their is equilibrium with as many coming off as going on
What can prevent G-actin nucleus from falling apart to speed up filament assembly?
Formins (FH2) with a ring structure that binds and stabilizes the nucleus, also Arp2/3 (actin related protein) that binds to existing nucleus site and build new filament that starts to form a network
What happens if you add actin nucleating proteins during filament assembly?
Eliminate most of the lag time
What is the concentration of G actin at steady state?
The critical concentration
What is the typical in vitro critical concentartion (Cc)?
0.1 uM
What happens when monomer concentration falls below Cc? And rises above?
F-actin begins to depolymerize, or polymerizes when higher
What does the actin filament prefer addition of? And what is it mostly comprised of?
Prefers to bind ATP bound G-actin, but because it is hydrolyzed most of the filament is comprised of ADP F-actin
Which end of the actin filament grows faster?
The positive end elongates 5-10X faster than the negative end because the negative end is stabilized by monomer heads, because there is a lower Cc at the positive end
In steady state where are subunits adding and subtractin on an actin filament? and what are the concentrations?
Adding to positive end and losing from negative end with length remaining constant and subunits treadmilling through, Cc- > G-actin concentration > Cc+
What proteins regulate actin polymerization (in vivo)?
Thymosin beta 4: sequesters ATP-G-actin and binds 1:1 blocking ATP site preventing polymerization
Profilin promotes F-actin assembly and binds 1:1 to bind opposite ATP binding cleft promoting filament assembly by allowing ADP exchange for ATP
How can actin be organized?
Into networks and bundles
Where are binding sites in bundles and networks?
In bundles they are close to eachother for support of structure but in networks they are far away to give more flexibility
What family do the actin cross-linking proteins belong?
Calponin homology-domain (CH-Domain) superfamily - have paired actin-binding domains
How do F-actin severing proteins promote polymerization?
They break filaments into fragments that allows for new site of polymerization
How does actin polymerization drive changes in cell morphology?
It give platelettes their shape, sends out projections in phagocytic cells, and actin can push membrane forward on flatten it out
Which actin binding Toxin binds to the positive end causing depolymerization?
Cytochalasin D
Which actin binding toxin binds to G-actin to prevent polymerization?
Latrunculin
Which actin-binding toxin prevent depolymerization by binding filaments?
Phalloidin - commonly used for labeling but it is very toxic to cells and causes they to become static
Where are intermediate filaments found?
In animal cells
What are IFs associated with and what is their main function?
Nuclear and plasma membrane to provide structure of mechanical support and organization and distribute tensile force
What are the 4 categories of IFs and where are they found?
Nuclear lamins found in nucleus
Keratins found in epithelium
Type III found in muscle, glial cells, neurons, astrocytes and fibroblasts
Neurofilaments found in mature neurons and developiong CNS
What is the most widely distributed IF?
Vimentin
What is the IF structure?
Dimers formed by coiled coil of two monomers with rod alpha helical core and globular C and N terminal domain, tetramers formed by two dimers in anti-parallel fashion or non polar in staggered arrangement, tetramers line up end to end to form protofilaments and then laterally to form protofibrils
What makes a mature IF?
4 protofibrils - symmetric and no polarity
Do IFs undergo exchange?
They are stable, but not static, subunit exchange occurs rapidly
How are IFs cross linked to one another and other cytoskeletal components?
IF associated proteins that organize the IF network, do not sever or cap IFs, do not sequester monomers, cause cross links of IFs to MTs (plectin)
How do IFs attach cells together?
They confer adhesiveness within and between tissues by hooking into junctions like desmosomes
What protein cannot form protofilaments and is a mouse model that has sever skin abnormalities?
Keratin, K14
Which filaments are dynamic and which are more stable?
IFs are stable and MF and MT are dynamic
What are the subunits of the filaments?
IFs = alpha helical rods and MF and MT are globular
Which filaments are nucleotide binding?
MF and MT, not IFs
What are the most rigid type of filaments?
Microtubules
What populations of cells are present in microtubules?
Stable long live cells like cilia/flagella, neuronal axons and unstable, short lived, cytosolic MT network or mitotic spindle
What is the structure of MTs?
Globular tubulin subunits arranged in a cylinder - tubulin subunits of heterodimer of alpha and beta tubulin monomers
What do tubulin dimers bind and what happens to them?
2 GTP molecules, the one on beta gets hydrolysed to GDP
How are tubulin dimers arranged?
Head to tail giving polarity where beta tubulin is the positive end (Where GDP is)
How do tubulin subunits align?
longitunally head to tail to form protofilaments, protofilaments associate with each other laterally to form cylinders, 13 protofilaments for a MT, bend around until they form a tube
How do MT lengthen?
Once protofilaments is formed into tube then MT lengthens by addition of GTP tubulin dimers - preferentially to positive end - hydrolyzed beta GDP receives a GTP cap to protect from dissociation during rapid growth
What does dissambly in MTs look like?
Dimers bent away from the tubule resulting in splayed or frayed ends - loss of GTP cap
Does disassembly or assembly proceed faster?
Disassembly
What is the kinetics of MTs?
Same as actin filaments - dimer concentration greater than Cc polymerization, dimer concentration less than Cc depolymerization, between Cc- and Cc+ is steady state
How does temperature effect MT stability?
Decreased temperature gives depolymerization, but increased temperature gives polymerization and is reversible
What do MAP MT proteins do?
Assemble and stabilize, crosslink to one another
What do Katanin and Op18 MT proteins do?
Destabilize by microtubule severing or binding tubulin dimers
Which drugs depolymerize or block dynamic changes to MTs and how?
Colchicine and colcemid - bind dimers and -prevent polymerization
Taxol binds and stabilizes MTs giving net increase in tubulin polymerization
Where is MT network centered/anchored? and where is it found in interphase and mitosis
The MTOC = Microtubule organizing center, basically the origin - found at centriole in interphase and at center of spindle formation in mitosis
What is the structure of the MTOC (aka centrosome)?
Pair of centrioles (9 triplet Mts arrange in pinwheel), MAPs to hold everything together and gamma (y) tubulin to nucleate tubule with negative ends of MTs oriented toward MTOC to give polarity
What is the arrangement of cilia and flagella?
9 + 2
What are the MT motor proteins and their classes? and what is their function?
Kinesin and dynein - provide intracellular transport of organelles (cytosolic for organelles, mitotic for spindles and chromosomes) via kinesin and dyneins, and beating of cilia and flagella via dyneins (axonemal)
What are the regions/structures of kinesin? and their functions? and what is the energy used during movement?
2 heads bind MT on beta tubulin every 8nm, neck determines direction, stalk, and tail region is variable and determine the cargo it carries - ATP hydrolysis moves the neck at each step
What does the mitotic apparatus look like during metaphase?
Comprised of the spindle and 2 asters with negative ends of MTs oriented towrad the centrosomes
What type of MTs attach to chromosomes during prophase?
Kinetochore MTs
What do the (-) end motors and (+) end motors do during prophase with MTs?
(-) end motors align polar MTs and (+) end motors separate centrosomes to establish spindle poles
What is the role of MTs in metaphase?
Chromosomes are positioned at the center of the spindle by motor proteins at both ends of kinetochore MTs
What is the role of MTs in anaphase?
MT motor and depolymerization of MTs right behind kinetochore are responsible for chromosomal transport toward the spindle poles
What is the role of MTs in telophase?
Actin/myosin contractile ring constricts the cell whereas the spindle MTs determine the position of the cleavage plane
What are some thinsgs that loss of cytoskeletal function can lead to?
COPD, infertility, neurodegenerative disease, cancer, liver cirrhosis, skin blistering
What is primary ciliary dyskinesia and what does it lead to?
non-functional or absent dynein arms results in ciliia or flagella that don’t beat can lead to COPD or infertility
