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

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• 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