Cell Bio- Exam 2

Slide Set 5: Vesicular Traffic, Secretion, and Endocytosis

Vesicular Transport

• Proteins are synthesized in the ER, then are moved from ER to golgi, once mature proteins are formed, they need to leave the ER (Secretory)
• After golgi, they have multiple different pathways
  • Constitutive secretion- constant secretion of proteins from cell, golgi to out of cell
  • regulated secretion- secretory vesicle takes protein out of cell from golgi
  • Endocytic- early endosome takes proteins from membrane to late endosome and then sometimes to lysosome
  • 
• Microscopy study with GFP
  • studied trafficking via GFP virus particles
  • use temperature, if temp inc, protein mvmt blocked
  • you can track proteins via fluorescent microscopy
  • results: there is trafficking within the cell, you can get a rough est of the time that it takes
  • tracking total fluorescence signal over time
• Oligosaccaride modification
  • mannose trimming occurs when oligosaccaride moves from ER to golgi
  • treated with endoglycosidase D which cleaves sugar from protein
• Vesicle Budding and Fusion
  • transport vesicle leaves donor compartment
  • transport vesicle fuses with target compartment
• Coated Vesicle Budding
  • SNARE protein helps transport vesicles recognize target membranes
  • membrane cargo protein and soluble cargo protein bind together
  • coat proteins surround vesicle
• Uncoated vesicle fusion
  • V SNARE proteins will interact with T SNARE proteins on membrane
  • Rabs protein- can help recognize which target mem they should fuse too, assists with docking

What is the mechanism by which vesicles are formed?

• Three types of coated vesicles
  • Clathrin coated - helps with transport from trans golgi network to late endosome and helps transports obj entering the cell via endocytosis
  • have heavy and light chains, as well as binding site for assembly particles
  • soccer ball structure
  • Functions:
    • help form mechanical force to form vesicle
    • coat subunits bind to surface of donor membrane
    • clathrin and other proteins help form bud/vesicle and help with the mechanical force of budding off
    • capture membrane receptors
    • clathrin and adaptin (bound together) bind to cargo receptor bound to cargo molecules in membrane, and then start budding process,
    • adaptin helps transmem receptor bind to coating proteins
      • certain aa are carried that signals adaptin to bind, these are then phosphorylated
  • Dynamin
    • required for pinching off of clathrin vesicles from donor membrane
    • polymerizes around the neck and then hydrolyzes GTP, conformational change initiated in dynamin that stretches vesicle neck until the vesicle pinches off
  • COP 1- in charge of moving protein from trans golgi back to ER
  • coatomer coated
  • intra golgi traffic, golgi to ER
  • ARF plays a role in coat formation
  • COP 2- helps with protein leaving ER to cis golgi
  • coatomer coated
    • Sar 1 uses COP 2 components
• GTPases
  • Active- when protein binds to GTP
  • GAP- hydrolyzes GTP to GDP
  • Sar 1 initially binds to GTP, then binds to Sec 12 to hydrolyze GTP, then recruits COP2 components to have GTP bound to mem
  • Sar 1- controls coat assembly on COP2 vesicles
  • inactive- off, GDP bound
  • GEF- releases GDP so GTP can be made
  • ARF- also a GTPase, plays role in coat formation in COP1 and Clathrin coated vesicles, intitially binds to GDP
    • 

What are the molecular signals on vesicles that cause them to bind only to the appropriate target membrane?

• SNARES and RAB GTPases play a role in vesicle traffic and fusion
  • generate tight interactions, help vesicles fuse to the donor membrane
• RAB GTPase
  • donor mem: RAB receptor, vesicle: RAB
  • mediate diff transport vesicles fused to diff transport membranes
  • many diff RABs in eukaryotic cells

How do transport vesicles and their target organelles fuse?

• SNARE and RAB help vesicle recognize donor membrane
• RAB will not help fuse, will help recognize membrane
• Vesicle Fusion Machinery
  • Vesicle Docking: V SNARE and T SNARE associate, RAB binds to RAB receptor
  • Assembly of SNARE complex:
  • SNAP 25- snare complex, includes V SNARE and Syntaxin
  • generates strong force to help fusion to the membrane
  • twisted very tightly together
  • Membrane Fusion
  • proteins work to untwist SNAP 25
  • fusion of membranes occurs
  • Disassembly of SNARE complexes
  • SNARE complexes disassociate and are free for another round of vesicle fusion, RAB also disassociates from the RAB effector

Steps in Secretory Pathway cont

• Vesicular Transport from ER to Golgi
• protein always goes from cis to trans face of golgi
• cis cisterna→ medial cisterna → trans cisterna
• ER retention signal- four aa, KDEL; if added at c term of protein it will return to ER from cis golgi bc it will bind to place on cis golgi and be recognized
• Cisternal progression through golgi glycosylation and other mods in golgi
  • removal of 3 mannose residues in cis golgi (-3 Man)
  • protein moves to medial golgi by cisternal maturation
  • 3 GlcNAc residues added , 2 more mannose removed, single fucose is added (+ 3 GlcNAc, -2Man, + Fucose)
  • processing completed in trans golgi by addition of 3 galactose residues and linkage of N-acetylneuraminic acid residue to each galactose (+3 Gal, + 3 NANA)
  • 
  • Role of glycosylation
  • post translational modification
    • helps protein become hydrophilic→ aids in folding
    • aid in transport (rarely- targeting to lysosome)
    • resistance to proteases (stability)
    • protein protein interactions
• Vesicular sorting at trans- golgi network
• Vesicular Trafficking to Final Destination (golgi to ___)
  • Endosome
  • Plasma Mem
  • constitutive secretion- unregulated membrane fusion
  • regulated secretion- regulated membrane fusion
  • Lysosome
  • some proteins go here
  • very acidic environment
  • v class pumps used with ATP to pump proton inside
  • lysosomes form a functional hub for cellular trrafficking pathways
    • ER→ Golgi→ lysosome
    • Pinocytosis→ lysosome
    • Phagocytosis→ lysosome
    • autophagy→ lysosome
  • How does cell know which proteins are sent to the lysosome?
    • M6P residues!
    • receptor on trans golgi network that will bind to M6P and will incorporate into vesicle and then will go to late endosome
    • if pH low in late endosome, M6P transferred to lysosome
  • Lysosomal Storage diseases
    • can be due to absence of 1 or more lysosomal hydrolases or the mistargeting of lysosomal hydrolases
    • characterized by tissue destruction or accumulation of undigested macromolecules
    • I cell- protein stuck in trans golgi, severe tissue destruction, GlcNac Deficiency
• Endocytosis
  • goes through plasma mem, through early endosome then late endosome, then lysosome
  • pinocytosis-
  • very tiny things; proteins, lipids. Goes through early, late, then lysosome
  • continuous process, rate depends on cell type
  • pinocytotic vesicle forms from clathrin coated pits in plasma mem
  • receptor mediated endocytosis- ligand binds to cell surface receptor, clathrin helps to form vesicle, clathrin coats vesicle
  • phagocytosis-
  • large things like bacteria; phagosome then to lysosome
  • feeding for lower single celled euks
  • multi celled orgs- used as a defense against invading microbes
  • requires surface receptors, triggered event
  • autophagy-
  • from ER, if we do not need certain organelles anymore, autophagosome forms then transported to lysosome
  • LDL Uptake
  • LDL- byproduct of fat transport, have ApoB protein
  • ApoB and LDL receptor bind
  • vesicle begins to form with help of clathrin coat
  • transported to early endosome→ late endosome→ lysosome
  • Disorders- LDL receptor missing, receptors do not associate with clathrin coat
  • Fate of cell surface receptors after endocytosis
  • recycling of receptor to same domain
    • receptor transported back to surface of membrane and pH will change→ receptor ready to bind to another LDL particle
  • degradation of receptor after endocytosis
    • in lysosome
  • transcytosis
    • any protein that is missent to basolateral side will be resent to apical membrane side
    • the vesicular transport of macromolecules from one side of a cell to the other

Slide Set 6: Microfilaments

The Cytoskeleton

• Functions of cytoskeleton
  • cell shape, mvmt, and contraction
  • organelle mvmt and organization
  • cell division
  • intracellular org and vesicle mvmt
  • interacting with signaling pathways
• basically like the bones of the cell
• Components
  • Microfilaments
  • actin filaments, thinner
  • Microtubules
  • tubulin dimers, thicker
  • Intermediate filaments
  • various, diff proteins combined together
• Cell signaling
  • signals tell cytoskeleton abt organization and mvmt of organelles as well as changes in cell shape, mvmt, and contraction

Actin Microfilaments

• Functions
  • org of intracellular organelles and transport of vesicles (myosin)
  • intracellular mobility (bacteria)
  • cellular stability
  • cellular motility
  • muscle contraction
• Lamellipodium
  • supported by growth of actin filaments, generates a protrusion structure to adhere to surface and move cell forward
• Polymerization and Dynamics
  • 1 actin filament= 2 strands
  • one + end (0.12 M), one - end (0.6)
  • g actin is monomer, microfilament polymer of actin
  • ATP binding cleft in actin structure
  • alpha, gamma, and beta actin: all associated with diff structures
  • G actin polymerization
  • g actin binds to f actin, elongating existing filament
  • can be added to + and - end, and leave from both sides
  • g actin dec to critical con→ polymer shrink
  • g actin inc above critical conc→ polymer inc in length
• Actin Binding Proteins
  • Polymerization- Profilin and Thymosin B4
  • Profilin- promotes polymerization
  • Thymosin b4- blocks polymerization of ATP
  • Length- Cofilin, Gelsolin
  • Nucleation and branching- Arp2/3
  • Crosslinking- Filamin
  • Motor Proteins- myosin
  • stability/cap end of filaments- capz and tropomodulin
  • CapZ- caps at + end
  • Tropomodulin- caps at - end
  • org of filaments/muscle contraction, binds to side of filaments- nebulin
• Actin based Motility
  • Formin - leads to assembly for long actin filaments
  • will form dimer structure
  • actin binds to structure and elongation commences
  • ARP2/3 complex can be used for polymerization to power motility, mediates branching
  • listeria monocytogenes uses actin polymerization to move through cells and from cell to cell→ hijack actin machinery and polymerize it to move around
  • ActA protein activates Arp2/3 to nucleate new filament assembly from preexisting filaments
  • filaments grow at + end until capped by Cap Z
  • actin recycled through cofilin, which enhances depolymerization at the - end of the filaments
  • this process propels bacterium forward
  • Toxins that perturb pool of actin monomers
  • Cytochalasin D- depolymerizes actin by blocking further addition of subunits
  • Latrunculin- inhibits g actin from adding to filament end
  • Jasplakinolide- stabilizes and binds actin dimers, lowers critical conc bar
  • Phalloidin- prevents actin filaments from depolymerizing by locking F subunits together
  • Actin also interacts with itself
  • types of lateral attachment of microfilaments to membranes
    • ankyrin- binds to Band 3 and then spectrin, forms network
    • band 4.1
• Actin Motor Proteins
  • myosin
  • can bind to actin and help generate contraction in muscle cells
  • composed of heavy chains and light chains, diff myosin has diff amts of each
  • myosin heads can bind to ATP and actin
  • Myosin 1
    • small, single head
    • step size 10-14nm
    • works with membrane association and endocytosis
  • Myosin 2
    • dimer (2 heavy chain)
    • 8nm step
    • bipolar filaments
    • works with contractions
  • Myosin 5
    • bigger, 2 heavy chains and more light chains
    • 36nm step
    • responsible for organelle transport
  • Myosin mvmt process
    • ATP binds to head grp, head group not associated with actin yet
    • ATP hydrolyzed, head grp rotated into position to bind, head grp binds to actin
    • power stroke occurs, Pi released and myosin straightened, moving actin filament left
    • ADP released, the ATP bound and head grp released from actin
  • Step size vs neck length
    • is myosin step size/velocity proportional to neck length?
    • YES, velocity inc with inc neck length
  • contractile ring
    • myosin 2 takes a large part in forming when cells are splitting, myosin 1 is on outside of cells
  • Sarcomere (not protein, just structure of skeletal muscle)
  • vertical component is Z band, in between is A band, myosin in between actin filaments
  • actin end facing inside is - end
  • sarcoplasmic reticulum- specialized region of the ER, regulates and stores Ca (Ca helps muscle cells to contract)
  • Cap Z- binds to + end of actin
  • Tropomodulin- binds to - end of actin
  • Nebulin- binds to side of actin filaments
  • Titin- binds to myosin and Z disk proteins
• Rho GTPases
  • membrane bound Rho proteins can bind effector proteins that cause changes in the actin cytoskeleton
  • dominant active rho- always keep making actin
  • Cdc42- filopodia formation
  • works at the front of cell, activates Rac
  • guys see a Rac and are activated
  • RacGTP- lamellipodia formation
  • leads to activation of Arp2/3 and Rho
  • RhoGTP- Stress fiber formation
  • leads to myosin 2 activation

Slide Set 7: Microtubules and Intermediate filaments

MIcrofilaments vs Microtubules vs Intermediate filaments

• microfilaments
  • actin binds ATP
  • form rigid gels, networks, and bundles
  • tracks for myosin
  • contractile machinery and network at cell cortex
• microtubules
  • tubulin binds GTP, rigid and not easily bent
  • trasks for kinesins and dyesins
  • organization for long range organelles
• Intermediate filaments
  • great tensile strength, less dynamic, unpolarized
  • no motors
  • cell and tissue integrity

Microtubules

• play a role in….
  • organization of organelles and transport of vesicles
  • mvmt of cilia and flagella
  • nerve cell, RBC, and flagellar structure
  • alignment and separation of chrom during mitosis
• Tubulin
  • monomer of microtubules, alpha and beta make up monomer
• Two populations of microtubules
  • Unstable short lived- assembles and disassembles rapidly
  • stable and long lived- remain polymerized for a long time (sperm flagella, RBC, nerve cells)
• Polymerization and Structure of Microtubules
  • Structure
  • tubulin has alpha and beta parts
  • bind to 2 GTP
  • alpha T GTP is never hydrolyzed
  • beta T GTP can be hydrolyzed
  • one end is beta T exposed→ + end
  • one end is alpha T exposed → - end
  • microtubules made up of 13 protofilaments → singlet
  • can have doublets (cilia/flagella) and triplets (basal bodies and centrioles) as well
  • Polymerization
  • microtubules assembled from MTOC
  • MTOC-any structure used by cells to nucleate and organized microtubules
    • centrosome falls into this category
    • neg end of microtubules at MTOC
  • gamma tubulin ring nucleates microtubule assembly
• Dynamics of Microtubules
  • Length over time: Assembly stage→ Catastrophe stage→ Disassembly stage→ Rescue Stage
  • Polymerization of tubulin into microtubules
  • protofilament first formed
  • alpha T first binds to protofilament, then beta T
  • sheet assembly
  • then form tube formation
  • GTP cap at top, GDP microtubule is the rest
    • GTP cap bc alpha and beta T carry GTP, addition of another Alpha and beta T will cause hydrolysis
    • \
      • end more smooth (assembly), - end more rough (disassembly)
  • Disassembly and reassembly of microtubules
  • cool to 4 deg, microtubule will disassemble
  • warm to 37 deg the microtubule will repolarize
  • Drugs that disrupt microtubule dynamics
  • colchincine- binds btwn alpha and beta T dimer so it cannot be used for polymerization, causes depolymerization
  • taxol- bind to side of tubules - stabilize the microtubule structure
• Binding Proteins
  • MAPs
  • can stabilize microtubules, similar to taxol
  • side binding
  • MAP2- longer
  • Tau- shorter
  • +TIPS
    • can regulate + end of microtubules
  • Motors
  • Kinesins
    • ferry cargo around the cell
    • ferry towards + end
    • have light chain, bind to ATP for energy resource
    • bind to microtubule with head groups, bind to vesicle via kinesin receptor
    • hydrolyze ATP to drive mvmt
    • Kinesin 1 and 2- organelle, mRNA, and chromosome transport
    • Kinesin 5- bipolar structure, 2 head grps, can bind to 2 diff microtubules, microtubule sliding
    • Kinesin 13- can regulate microtubule end disassembly
    • Process
    • first head group, (leading head), no ATP, bound to microtubule
    • leading head then binds to ATP
    • conformational change induced, following head swings forwards
    • following head becomes leading head
    • new leading head releases ADP which it was originally bound to, and new following head hydrolyzes ATP to ADP and then process restarts
  • Dyneins
    • ferry cargo around the cell towards - end
    • Power stroke of dynein- ATP hydrolysis causes change in orientation of head→ mvmt of MT
    • dynactin- bind cargo, make dynein more processive
    • LIS1 protein- interact with ATPase domain of dynein to elongate power stroke

Intermediate filaments

• heterogeneous
• great tensile strength
• no known motors use them as tracks
• more stable than filaments or tubules
• no intrinsic polarity
• made up of protofilaments that can form diff structures
  • have N term and c term and head and tail end
• keratin, lamin, vimentin

\