Test 2 Q - Answers
1. Actin filaments form (like "tinkertoys") from actin monomers - assesmbling on their assembly (+) end (torwards the cell membrane) and where ATP cap is in equilibrium filament and disassembling as needed on their disassembly (-) end (ADP cap end in equilibrium). Don't come apart because the membrane has an excess of lipids that have lateral motion around the membrane. When actin pushes out aganist the membrane, the membrane phospholipids can pull apart from one another somewhat (although not apart enough to overcome the Van der Waals and hydrophobic attractions that keep the membran intact) forming spaces between lipids that the excess lipids from other areas of the membrane can move into.
2. Compare and contrast Actin, Microtubules, and Intermediate Tubules. Be complete - use all the information I told you in class.
Actin:
Structure / Polarity - "donut" shaped alpha, beta, gamma monomers 42 kD, ~375 AA's differ from one another by 4 -5 AA's (?!?!), each has a pocket containing ATP/ADP. Polarity of each mono. gives filament polarity. ATP and ADP caps ends, 6 nm in diameter filaments.
Assembly / Disassembly - ATP cap is ass end, ADP cap w/ less binding NRG is diass end. At eq. 1 mono added for each taked - gives treadmilling in vitro and vivo
In vitro conditions - will ass / diass in vitro and in vivo. Know what's needed for this...
Nucleation cites - actin forms at nuc. sites at Memb Anchor proteins just inside PM.
Intracellular prefs - cell. conditions - treadmillling occurs as actin filaments are assembled as "tinkerotys" to extend cell fila. and lamelli. don't bk PM b/c extra lipids in memb. come to fill spaces when actin pushes out
Assoc. Proteins - Myosin I, Myosin II - light and heavy chains (draw a ceduceus) 220 kD each heavy, 22 kD each light chain. Bundled to form "bouquets" of fibers layered between bands of actin, and are Ca 2+ dependent activated to contract muscles. (Chk this). Villin - first thought to be actin bunding protein in brush barrier cells, came out at 72 kD on SDS-PAGE. BUT Villin works based on [] of free Ca 2+. Fimbrin at 68 kD turned out to be the bundling protein. Capping proteins (?!?!?!) - let cell control start and stop of creation of actin filaments (?!?!?).
MTs:
Structure / Polarity - each TUBULIN monomer is actually a homodimer? with alpha and beta subunits, each 55 kD and ~ 450 AA's in length. These polymerize together in a "braided" chain - end to end the chain of monomers forms a protofilament. as they braid around to form a MT - they form 13 alternating subunits around an open space (draw better) than is 25 nm on its outer cirum and 15 nm on the inner, mono. ring is ~ 5 nm in width. The units each pocket a GTP/GDP - only the beta subunit transforms to GDP. Monomers and hence the filament have polarity.
Assembly / Disassembly - have + and - ends, disintigrate rapidly in cell.
In vitro conditions - MTs treadmill in vitro but NOT in the cell. - extracted from cow brains, What are conditions in vitro - Know....
Nucleation cites - Centrosome - don't touch the centrioles, but will not form witout centriols in animal cells??!?!? Have a "cap" ?!?!?! inside the centrosome- this is the (-) end. (+) ass. end out into cytoplasm from centrosome center.
Intracellular prefs - do NOT treadmill in cell - disintegrate too rapidly. Grow out to near PM. Act as "roads to Rome" of the cell.
Assoc. Proteins - Dynein connects like legs outside of vesicles / organelles to walk along MT to (-) Kinesin - same, but out to (+) part of MTs.
IF's:
Structure / Polarity - Monomeric units consist of 4 strands of peptides that alternate btw 5 linear and 4 alpha helical regions. Hydophobic areas (at the linear parts?) cause the strands to bond more tightly than almost another natural substance in animal cells. b/c of alternating "lines of fracture" - IF's are "brick shithouses" of natural fibers. Each of 4 strands is polar with N and C term ends. Brought together in 4 they "lose" polarity, however.
Assembly / Disassembly - 4 strands come together w/ hydrophobic regions, DO NOT disass.
In vitro conditions - have to boil at 100 deg C in 2% SDS and 4 M Urea to denature.
Nucleation cites - right at Nuclear membrane - stands extrude through pores to outside and bond immeidately to outside of nuclear memb. Transported back in thru pores to form Nuc. lamins. This forms with "stainless steel jockstrap" springy cage that protects the Nuc. from crushing.
Intracellular prefs - 4 strand monomers twist together to form long strands that then bundle in 4 cables which circle around in 8 bundles to form the IF.
Assoc. Proteins - we didn't cover yet? Not used to transport - no polarity!!!!!
3. ATP, GTP, CTP, TTP. ATP is found in actin, GTP in microtubules. It is not known exactly why actin filaments use ATP in particular for a non-NRG generating purpose, but ATP gets converted into ADP by an enzyme. These conversions, happening randomly, eventually form the ATP and ADP caps - the ATP is the "assembly end", and ADP the disassembly end (the ADP has 1 less negative charge and dissociates more readily).
In MT's, in the Tubulin monomer (actually a heterodimer), both the (joined) alpha and beta subunits start off containing GTP, but only the beta subunit GTP gets converted to GDP. More needed ...
3. What are some examples of nucleotide tri-phosphates? Where specifically to we find the ones we talked about in class? How are they used?
ATP, GTP CTP, TTP. ATP is used in actin filaments, not as NRG source, but to mark the filament for ass / disass ends. GTP is found in tubulin monomers of MT's. It also signifies polarity of the MT.
4. What is polarity when in biological macromolecules? Why is it dissimilar to electrical charge? What causes polarity?
As in class - polarity means different directionality. It is not electrical charge b/c doen't connote free electrons moving in flow from one place to another, but is the position change of monomers as the cycle through an actin filiment. Or the directionality that vesicles are "walked" along a cytoskelatal fiber to move throughout a cell. This only happens in one direction for a given molecular motor. Polarity or lack of it arise from the polarity of monomers.
5. What are some of the "families" seen in cellular matix systems? What are some of the members? How are the families organized?
Actin filaments have alpha, beta, gamma types, but only on family member makes up a single actin filament (?!?!?). In contrast, in tubulin, alpha and beta units are attached together to make a monomer - so there is intermingling of familial types.
6. What makes IF's the "brick shithouses" of the cell biology world?
The monomers align in a staggered conformation to form a filament, much like brick walls are staggered. This non-grid alignment means that there are no single potential lines of fracture directly across or along the filament, making IF's even more strong. Additionally, there are hydroprobic strips every 7 repeats (?!?!?!) that make the assembled IF extermely strong.
7. How can we verify the polarity of MT's in the cell?
In vitro, can use free monomers which under cell cond. will form protofilament "wings" off the MT. Through thin-slice EM ALONG the MT, these were also seen. Turns out CW is to (-) end - to the centrosome. CCW is to (+) end - to PM.
8. How did scientists first test how myosin works in cells? What steps did they do to test this?
2. Compare and contrast Actin, Microtubules, and Intermediate Tubules. Be complete - use all the information I told you in class.
Actin:
Structure / Polarity - "donut" shaped alpha, beta, gamma monomers 42 kD, ~375 AA's differ from one another by 4 -5 AA's (?!?!), each has a pocket containing ATP/ADP. Polarity of each mono. gives filament polarity. ATP and ADP caps ends, 6 nm in diameter filaments.
Assembly / Disassembly - ATP cap is ass end, ADP cap w/ less binding NRG is diass end. At eq. 1 mono added for each taked - gives treadmilling in vitro and vivo
In vitro conditions - will ass / diass in vitro and in vivo. Know what's needed for this...
Nucleation cites - actin forms at nuc. sites at Memb Anchor proteins just inside PM.
Intracellular prefs - cell. conditions - treadmillling occurs as actin filaments are assembled as "tinkerotys" to extend cell fila. and lamelli. don't bk PM b/c extra lipids in memb. come to fill spaces when actin pushes out
Assoc. Proteins - Myosin I, Myosin II - light and heavy chains (draw a ceduceus) 220 kD each heavy, 22 kD each light chain. Bundled to form "bouquets" of fibers layered between bands of actin, and are Ca 2+ dependent activated to contract muscles. (Chk this). Villin - first thought to be actin bunding protein in brush barrier cells, came out at 72 kD on SDS-PAGE. BUT Villin works based on [] of free Ca 2+. Fimbrin at 68 kD turned out to be the bundling protein. Capping proteins (?!?!?!) - let cell control start and stop of creation of actin filaments (?!?!?).
MTs:
Structure / Polarity - each TUBULIN monomer is actually a homodimer? with alpha and beta subunits, each 55 kD and ~ 450 AA's in length. These polymerize together in a "braided" chain - end to end the chain of monomers forms a protofilament. as they braid around to form a MT - they form 13 alternating subunits around an open space (draw better) than is 25 nm on its outer cirum and 15 nm on the inner, mono. ring is ~ 5 nm in width. The units each pocket a GTP/GDP - only the beta subunit transforms to GDP. Monomers and hence the filament have polarity.
Assembly / Disassembly - have + and - ends, disintigrate rapidly in cell.
In vitro conditions - MTs treadmill in vitro but NOT in the cell. - extracted from cow brains, What are conditions in vitro - Know....
Nucleation cites - Centrosome - don't touch the centrioles, but will not form witout centriols in animal cells??!?!? Have a "cap" ?!?!?! inside the centrosome- this is the (-) end. (+) ass. end out into cytoplasm from centrosome center.
Intracellular prefs - do NOT treadmill in cell - disintegrate too rapidly. Grow out to near PM. Act as "roads to Rome" of the cell.
Assoc. Proteins - Dynein connects like legs outside of vesicles / organelles to walk along MT to (-) Kinesin - same, but out to (+) part of MTs.
IF's:
Structure / Polarity - Monomeric units consist of 4 strands of peptides that alternate btw 5 linear and 4 alpha helical regions. Hydophobic areas (at the linear parts?) cause the strands to bond more tightly than almost another natural substance in animal cells. b/c of alternating "lines of fracture" - IF's are "brick shithouses" of natural fibers. Each of 4 strands is polar with N and C term ends. Brought together in 4 they "lose" polarity, however.
Assembly / Disassembly - 4 strands come together w/ hydrophobic regions, DO NOT disass.
In vitro conditions - have to boil at 100 deg C in 2% SDS and 4 M Urea to denature.
Nucleation cites - right at Nuclear membrane - stands extrude through pores to outside and bond immeidately to outside of nuclear memb. Transported back in thru pores to form Nuc. lamins. This forms with "stainless steel jockstrap" springy cage that protects the Nuc. from crushing.
Intracellular prefs - 4 strand monomers twist together to form long strands that then bundle in 4 cables which circle around in 8 bundles to form the IF.
Assoc. Proteins - we didn't cover yet? Not used to transport - no polarity!!!!!
3. ATP, GTP, CTP, TTP. ATP is found in actin, GTP in microtubules. It is not known exactly why actin filaments use ATP in particular for a non-NRG generating purpose, but ATP gets converted into ADP by an enzyme. These conversions, happening randomly, eventually form the ATP and ADP caps - the ATP is the "assembly end", and ADP the disassembly end (the ADP has 1 less negative charge and dissociates more readily).
In MT's, in the Tubulin monomer (actually a heterodimer), both the (joined) alpha and beta subunits start off containing GTP, but only the beta subunit GTP gets converted to GDP. More needed ...
3. What are some examples of nucleotide tri-phosphates? Where specifically to we find the ones we talked about in class? How are they used?
ATP, GTP CTP, TTP. ATP is used in actin filaments, not as NRG source, but to mark the filament for ass / disass ends. GTP is found in tubulin monomers of MT's. It also signifies polarity of the MT.
4. What is polarity when in biological macromolecules? Why is it dissimilar to electrical charge? What causes polarity?
As in class - polarity means different directionality. It is not electrical charge b/c doen't connote free electrons moving in flow from one place to another, but is the position change of monomers as the cycle through an actin filiment. Or the directionality that vesicles are "walked" along a cytoskelatal fiber to move throughout a cell. This only happens in one direction for a given molecular motor. Polarity or lack of it arise from the polarity of monomers.
5. What are some of the "families" seen in cellular matix systems? What are some of the members? How are the families organized?
Actin filaments have alpha, beta, gamma types, but only on family member makes up a single actin filament (?!?!?). In contrast, in tubulin, alpha and beta units are attached together to make a monomer - so there is intermingling of familial types.
6. What makes IF's the "brick shithouses" of the cell biology world?
The monomers align in a staggered conformation to form a filament, much like brick walls are staggered. This non-grid alignment means that there are no single potential lines of fracture directly across or along the filament, making IF's even more strong. Additionally, there are hydroprobic strips every 7 repeats (?!?!?!) that make the assembled IF extermely strong.
7. How can we verify the polarity of MT's in the cell?
In vitro, can use free monomers which under cell cond. will form protofilament "wings" off the MT. Through thin-slice EM ALONG the MT, these were also seen. Turns out CW is to (-) end - to the centrosome. CCW is to (+) end - to PM.
8. How did scientists first test how myosin works in cells? What steps did they do to test this?
posted by Ari at 3:39 PM
<< Home