D103 MT Review

How does dysfunction of organization affect the cell?

Leads to disease

Ex) centrosome, golgi membrane, ER network, mitochondria

Model Systems

Simple (easy, cheap, quick)

Must have conserved mechanism

Availability of genetics (ie genome sequenced)

Light microscopy limitation?

Resolving power is ½ wavelength of source light, need shorter light to see smaller molecules (ex electron microscopy)

General phospholipid structure

Glycerol backbone with diff head group and tails

Lipid distribution in a membrane

Asymmetrical, can get curvature bc of smaller head sizes

Mobility of phospholipids

Addition of heat leads to a more fluid membrane

Regulation of membrane fluidity

Length of chains → shorter chains get less interaction, more fluid membrane

Saturation degree → highly saturated = more kinks, more fluid

Cholesterol levels → stiffens membrane and increases thickness, less fluid

Transmembrane proteins

Must have hydrophobic domains that are inserted into the membrane

Hydropathy plot

Top area refers to hydrophobicity, number of peaks surpassing 20-25 residues coincide with a trans membrane domain

Membrane transport proteins

All have transmembrane domains

Allow water soluble membranes to pass through

Undergo a conformational change

Transporters: Uniporter

Passive transport down a gradient

Transporters: Symporter

Moves two molecules at once, movement of one particle down its gradient provides energy to move another particle against its gradient in the same direction

Transporters: Antiporter

Moves two molecules at once in diff directions, energy gained from movement of one particle down its gradient

Pumps

Use ATP to move a molecule up its gradient

Ion channels

Passive transport of an ION down its gradient, opens due to binding of some ligand

Vmax

Each protein is working at max rate

Trans-epithelial transport of glucose

Na+/Glu symporter takes advantage of the low Na+ concentration in the lumen to move glucose

This low Na+ concentration is maintained by a Na+/K+ pump on the blood vessel membrane

Glucose then moves to blood by a simple uniporter

Role of mitochondria

Takes up food, breaks it down into energy (ATP) through ox phos

Oxidative phosphorylation

Food from cytosol → moves into inner membrane → acetyl-CoA → TCA makes electron carriers → ETC (on inner membrane) → generates proton gradient → ATP synthase (inner mito membrane) uses gradient to make ATP

UCP (uncoupling protein)

Uncouples proton gradient from ATP synthase

Forms an alternative pathway for protons back into the mito matrix

Releases energy as heat

Key features of mitochondria

Double membrane

Abundant (proportional to energy req), distributed throughout the cell

Dynamic as they regularly fuse and divide as they move around the cell

Inner mitochondrial membrane

Many cristae to increase surface area

High protein content

Has a phospholipid with 4 tails → EXTREMELY hydrophobic an robust (impermeable)

Outer mitochondrial membrane

Has porins, permeable to small molecules

mtDNA

Mitochondria have their own DNA that is transcribed/translated on demand but in house machinery

Most mito proteins are still encoded by nuclear DNA

Dense packing/no introns

No proof-reading/repair so mutation rate is really high

Passed down from mother (mtDNA is not evenly distributed into daughter cells so can have diff levels of mutational severity)

Immunofluorescent staining

Primary antibody: inject whatever protein into a host that will make antibodies for it

• must have diff animals if staining multiple things

Secondary antibody: inject primary into ANOTHER animal, it will recognize the animal antibody, has a fluorescent light

Cytoskeletal elements

Microfilament actin (F-actin)

Intermediate filament

Microtubules (tubulin)

V dynamic due to monomer construction and regulatory proteins that control de/polymerization

Fluorescent tagging

Tag genes with GFP or fluorescent marker

Then transcription/translation results in protein that glows

F-actin

Cell shape and migration

Made of G-actin subunits

Assembly depends on ATP hydrolysis (reqs energy)

Has + and - end

Nucleation: monomers slowly form stable complexes

Cytosolic concentration of G-actin determines growth/shrinkage

Above Cc = growth

Below Cc = shrinkage

• Cc is different for +/- end, so growth rates are different

• Addition is faster at + end

Treadmilling

Cc- > Ccyto > Cc+

+ end continuously grows (to reach Ccyto) while - end continuously shrinks (to reach Ccyto)

Cell maintains the same filament content

Very dynamic behavior lends to flexibility → cell can change to make quick responses to diff signals

Regulated by accessory proteins

• Sequestering proteins and G-actin activators: regulate concentration of available G-actin (neg/pos)

• capping proteins: protest from depolymerization

• Nucleators: nuclear assembly or linear and branched actin

Actin coordinated cell migration

1. Protrusions form at leading edge due to polymerization

2. Atttach to substratum

3. Cell body moves using myosin generated force

Aka polymerization, adhesion, and translocation

Small GTPases

Rho (stress fiber and contraction), Rac (protrusion formation), and Cdc42 (morphology for metastasis)

All regulate cell migration through actin → do so by regulating downstream effectors

They themselves are regulated by GEF and GAP

• GEF activates the protein by phosphorylation to GTP bound state

• GAP deactivates protein by dephosphorylation to GDP state

Myosin

Actin-based motor proteins

Two binding sites: ATP (for energy) and actin

Basic unit = sarcomere

Actin + end is capped, - end is held by myosin

Ca++ binds to troponin, allowing tropomyosin to lift away and reveal myosin binding site for ATP

Myosin then “walks” to + end to contract

Tubulin

organize organelles and mediate intracellular transport

composed of heterodimers

• alpha is GTP bound

• beta is GDP bound but can be exchanged with soluble GTP

elongation occurs at + end

GTP cap refers to GTP accumulation at the + end

• rate of hydrolysis from GTP to GDP is slower than addition of dimer subunits

Tubulin dynamics

catastrophe refers to accidental loss of GTP cap, leads to rapid shrinkage

rescue refers to regain of GTP cap and rapid growth

cap loss/gain controlled by local GTP-bound tubulin concentration

Structural consequences of GTP hydrolysis

loss of GTP cap

curling of microtubule filaments

MTOC

microtubule organizing center

where mts grow from, provide a nucleation site

during interphase, centrosome is the main MTOC

nucleating sites are gamma-tubulin rings, grow from plus end while minus end is capped

Cargo transport with microtubules

kinesin carries cargo to the plus end (K+ like potassium)

dynein moves cargo to the minus end

• has two head domains: one for ATP and one for the mt

• movement requires ATP hydrolysis

organelle membranes contain motor receptors to determine which motors can bind and deposit their cargo where

MT during mitosis

significant increase in MT nucleation during mitosis

more dynamic bc they have to search for chromosomes (do so by continuously reaching out (growing/shrinking)

FRAP (fluorescence recovery after photobleaching)

expression GFP fusion protein, photo bleach, determine how fast GFP signal recovers

premise is that the more dynamic neighboring cells are, the faster they move to fill up that hole and the faster the signal recovers

Taxol

binds to mts and inhibits mt depolymerization → inhibits dynamics and thus halts the process of mitosis

kills of hyper prolific cancer cells

Intermediate filament

gives cell mechanical strength

ex keratin in epithelia or nuclear lamins

polymer formation is spontaneous, however depolymerization requires energy

Lamins

provide structural support for cell

contain nuclear localization agent NLS

Nuclear envelope during mitosis

must breakdown

requires energy to dissemble lamina

regulated by kinase induced phosphorylation

Keratin

a type of intermediate filament in the epithelial cells that connect neighboring tissue

Desmosome

a cadherin-keratin structure that links IF with neighboring cells and ECM

• cadherin and keratin also require an anchor protein to mediate their connection

cell to cell

Cadherin

Ca++ dependent adhesion

have Ca++ binding sites, when bound they straighten out the structure and mediate cell to cell connection

cells that express the same cadherin turn to interact with each other

Cadherin and cancer

dysregulation frequently associated with cancer

loss of cadherin leads to increased migration and invasion of cells

epithelial cells undergo EMT and metastasize

Hemidesmosome

cell junction that anchors IF to the basal lamina

cell to ECM

Adherens junction

cadherin links an actin bundle to another bundle in a neighboring cell

cell to cell

Focal adhesion

uses integrins (alpha and beta subunit) to link the cytoskeleton (actin and IF) to the ECM

• binding mediated by adaptor proteins

cell to ECM

Integrins

a transmembrane protein used to link cytoskeleton to ECM

binding of the ligand causes conformational change

• outside-in activation: ECM ligand binds to activate integrin and thus intracellular events

• inside-out activation: actin associated protein binding activates integrins and then extracellular events

inactive form is compact, active form is stretched out

Actin polymerization

induces integrin activation and focal adhesion

ex of inside-out activation

ECM

collection of extracellular molecules that provide structural/biochemical support for surrounding cells

contains insoluble fibers such as collagen/elastin/etc

Collagen

extremely important ECM component

vitamin c is a cofactor for collagen processing

Principles for protein transport

has to have a sorting signal

signal has to get recognized by a specific receptor

receptor should bring protein to translocation machinery

transported protein gets released

energy input for transport

processing required for function

Sorting signal

must be necessary and sufficient

may be removed upon arrival

signal sequences or patch (which is made of distant aa residues that make a patch when processed)

Protein transport into nucleus

nuclear pores constitutively open, but require NLS

importin proteins bind to the cargo when they recognize NLS

move into nucleus where Ran GTPase binds to importin, leaving cargo

• GTPase activated by GEF in nucleus

Ran GTPase and importin move out through nuclear pore to cytosol, where GAP converts it to Ran GDPase and releases importin

Import into mitochondrial matrix

sorting signal is amphipathic helix

proteins imported in unfolded state, require chaperones

transport machinery TOM (outer) and TIM (inner)

driving force for import is ATP and a proton gradient

ER

stores Ca++ for muscle contraction

receives, modifies, and transports proteins

synthesizes lipids

• smooth: vesicle formation

• rough: protein import, secretory pathway, folding processed here

Protein import to ER

SRP or signal recognition particle is recognized by SRP receptor

translocated by translocons

GTP hydrolysis powers translocation

Translocation of soluble molecule

ER signal sequence is read then cleaved by signal peptidase after translocation

goes straight through

Translocation of transmembrane molecule

ER signal sequences is read then once the stop-transfer sequence is read, the protein stops moving through the membrane and the signal sequence is cleaved

Topology of ER transmembrane protein

C-terminal in cytosol

N-terminal in cytosol

ER proteins get glycosylated when they enter the ER

N-linked: initiated in ER, also in golgi

O-linked: only in the golgi

both just add a sugar tree to the protein

ER proteins are associated with chaperones

prevent premature folding

v strong bond to protein, requires energy to break

chaperones also recognize incorrect glycosylation

Folding issues and UPR

glucosidases remove sugar tree

if folded incorrectly, exit the ER

if not, another protein adds the tree back

misfolded proteins leads to ER stress

• UPR tries to solve the issue by halting protein prod, influx, and increasing chaperones

• when the cell gives up, it will undergo ERAD to degrade misfolded proteins and induce apoptosis

ERAD

when misfolded protein gets pulled out of ER and then degraded in proteosome

Secretory pathway

mediated by vesicles

• clathrin: trans-golgi network, endocytosis

• COPI: golgi to golgi, golgi to ER

• COPII: ER to golgi

divided into early and late stages

Proteins involved with vesicle budding/trafficking

assemble GTPase → coat assembly from donor compartment

Rab GTPase → vesicle docking

SNARE → mediate fusion with target compartment

Vesicle formation

small GTPase recruited to donor membrane

inner and outer coat proteins recruited

coat proteins selectively bind and recruit cargo

vesicle buds

Vesicle fusion

vesicle gets uncoated to expose v-SNARE proteins

Rab initiates docking by finding an effector on the target

v-SNARE binds to t-SNARE

vesicle fuses to target compartment

• SNARE proteins get hydrolyzed and then recycled

Botox

inhibits muscle contraction by blocking synaptic vesicle fusion

• bind and cleave SNARE proteins

Early secretory pathway

ER to cis-golgi (COPII)

Cis-golgi to ER and golgi to golgi (COPI)

Late secretory pathway

proteins are modified in golgi

Protein trafficking between ER and golgi

forward is mediated by COPII

backward is mediated by COPI

• for retrieving recycled proteins

• maintaining membrane balance

• missorted ER proteins

KDEL

protein retrieval signal

KDEL receptors are super pH sensitive, in the golgi it will have high affinity for KDEL and help move it back to the ER

Constitutive secretion

default, always on

for proteins in PM and ECM

Regulatory secretion

stored, wait for signal stimulation

Lysosome

major function is to eat/digest things with its many enzymes

most efficient at low pH maintained by proton pump

clathrin vesicles transport cargo to lysosome

Vesicle transport to lysosome

M6P sorting signal

• pH sensitive, dissociates in the more acidic lysosome environment

clathrin vesicle buds, congregate to form early endosome which congregate with each other to form late endosome

fusion

Protein modification

only permanent one is lipid/GPI anchor

Ubiquitination

any protein containing a lysine residue can be modified

ubiquitin also has a lysine, can bind to more of itself for polyubiquitination

• the more you add the greater the mw

three steps mediated by E1-3

• DUB also removes ubiquitin

Polyubiquitination

targets proteins for degradation through proteasome

modify in cytosol

Phosphorylation

modification of Ser, Thr, Tyr

adds negative charge