CELL BIOLOGY EXAM #3

microtubules

microtubules

-long stiff, cylindrical structure composed of the protein tubulin.

-Used by eukaryotic cells to organize their cytoplasm and guide intracellular transport of macromolecules and organelles.

-They also segregate chromosomes during mitosis.

-a main structural component of eukaryotic cilia and flagella

Microtubule structure

-structurally distinct ends → polarity - minus end(alpha-tubulin) and plus end(beta-tubulin)

-subunits consist of a dimer of alpha and beta tubulin that stack into a protofilament

-13 protofilaments join to make a hollow tube

Microtubule polymerization

microtubules are nucleated at the centrosome, gamma tubulin rings act as the nucleation sites. Minus ends are at the centrosome and the plus ends radiate outwards

Microtubule functions

1.) Divide chromosomes during mitosis and meiosis

2.) move cargo around cells ( vesicles, organelles) → use motors

3.) cilia/flagella movement

dynamic instability

-microtubules can rapidly switch between growing and shrinking (VERY important for cell function)

-1/2 tubulin is free in cytosol, 1/2 in microtubules

-General Process

• Tubulin dimers bind to GTP and add to the plus end, form a GTP cap

• if polymerization is fast, GTP tubulin is being added faster then hydrolysis can occur, if hydrolysis and the last GTP is hydrolyzed then it tips towards disassembly

Microtubule Accessory Proteins ( MAPs)

-change the function or dynamics of microtubules

-capping proteins→ stabilize plus end

-others can cleave or make an end less stable

Microtubules in Mitosis + Meiosis

-attach to sister molecules during prometaphase and pulls sister chromatids apart in anaphase

Microtubules → Movement of Cargo

-act as tracks for transporting cargo

-motor proteins drive movement → kinesins walk towards the plus ends and dyneins towards minus end

-structure

• two globular heads that walk along the beta subunits and the tail binds to cargo

Movement of Cargo → process

1.) ADP bound head binds to microtubule, the trailing head is bound to ATP

2.) trailing head hydrolyzes ATP and binds to microtubule

3.) leading head exchanges ADP for ATP and whip around the trailing head which now binds to the microtubule

-Cycle so it Repeats-

Cilia + Flagella

-cilia move fluid or propel the cell through its environment

-Microtubules are arranged in 9+2 array ( 9 doublets surrounded 2 central single)

cytoskeleton

an intricate network of protein filaments that extends throughout the cytoplasm

supports cytoplasm volume, highly dynamic and continously reorganized

framework is composed of three protein types → filaments, microtubuoles + actin

intermediate filaments

main function is to enable cells to withstand mechanical stress from stretching - tensile strength

found in the cytosol of animal cells and nucleus of all eukaryotic cells

general intermediate filament structure

strong + ropelike

strands are composed of fibrous subunits that contain a centralized rod domain with distinct unstructured domains at either end

do not have direction → motor proteins do not walk on them

rod domains

extended alpha helical region that forms dimers in a coiled-coil configuration and dimers run in opposite directions to form a staggered tetramer ( soluble subunits)

central rod domains of different filaments are all similar in size and amino acid sequence ( terminal head + tail domains vary)

intermediate filament classes

keratin filaments, vimentin + vimentin-related filaments, neurofilaments, nuclear lamins

keratin filaments

in epithelial cells - cytoplasm

most diverse class and typically span the interior of cells (ends connected to desmosomes)

distribute stress when the skin is stretched

ex. epidermal layer of skin, hair, feathers, claws

vimentin + vimentin-related filaments

in connective- tissue cells, muscle cells, support cells (cytoplasm)

neurofilaments

nerve cells - cytoplasm

found along the axons of vertebrate neurons - provide strength and stability

nuclear lamins

underlie and support the nuclear envelope

nuclear envelope structure

-supported by a meshwork of intermediate filaments

-2D meshwork formed by nuclear lamins

-disassembles and reforms during each cell division

-controlled phosphorylation and dephosphorylation

-linker proteins connect cytoskeletal filaments and bridge the nuclear envelope

phosphorylation and dephosphorylation of lamins

• phosphorylation of lamins weakens the interactions between tetramers causing filaments to fall apart

  • protein kinase

• dephosphorylation allows the lamins to reassemble

  • protein phosphates

Actin

-helps cells move and adopt different shapes

-Examples

• Microvilli in intestines - increase surface area

• contractile bundles

• pseudopods at leading edge of the crawling cell

• contractile ring to separate dividing cells -

Actin Structure

-shorter and more flexible then other filaments

-they can be bundled

-have polarity → plus and minus ends

Treadmilling

-subunits are added quiclky to the plus end and are more likely to be lost from the minus end

-Actin subunits bind to ATP → more stable at plus end

-once ATP hydrolyzes to ADP the subunit isnt stably bound

Acting Binding Proteins

help regulate actin dynamics and functions

Adherens Junctions

-structures used to connect actin in two cells to another

-linker proteins connect Actin in the cytoplasm to transmembrane cadherin proteins

-actin can contract which causes the epithelial sheet to change shapes

Actin Polymerization Regulation

-Arp 2/3 complex nucleates new branching actin filaments

• can’t form filaments but the plus end looks like actin and can start a new filament

- When the activating factor is added, Arp2/3 bind in a conformation that is like the plus end and actin can assemble on the end

Actin Functions

-Cell crawling

-myosin motors

-muscle contraction

cell crawling

Actin is concentrated in a layer beneath plasma membrane - CELL CORTEX

1.) Cell pushes out protrusion at leading edge - actin does pushing

2.) Protrusion uses focal adhesion - contacts to adhere to surface

3.) rest of the cell drags its self forward - myosin motors and depolymerize actin at cell rear

-the plus end of action points to the leading edge of the cell - actin meshwork

myosin motors

-has a single head and is actin-dependent, needs ATP for movement. moves from the minus end to the plus end, tail determines the cargo

-General Process

• myosin is attached to actin, ATP binds to myosin and releases myosin in order to hydrolyze ATP which recocks the head and a Powerstroke occurs when ADP is released

  • rigor mortis occurs if there is no ATP to release myosin from actin

muscle contractions

-muscle cells are multinucleate + form one big muscle cell

-plus end of actin is anchored to a Z disc and the myosin walks toward the disc causing contractions -shortens

-regulation → nerves trigger action potential and Ca2+ is released. Tropomyoisin binds to actin and prevents myosin binding to actin ( absence of Ca2+). When Ca2+ concentration is high, a conformational change is induced which allows myosin to bind to actin - muscle contraction

signal transduction

the process of cell communication with each other and their environment

General Signal Transduction Concepts

-the same signal can affect different cells differently

-signaling can be fast or slow → altering function (fast) and new protein synthesis ( slow)

-Signaling can be regulated by both positive and negative feedback → responses like a switch or all-or-none response ( positive) or pathway shutdown (negative

6 Steps of a Signaling Pathway

1.) Synthesis and release of signaling molecule by signaling cell

2.) Transport of signal to target cell

3.) detection of the signal by a specific receptor protein

4.) intracellular signaling molecules transfer signals from receptors at the plasma membrane to target protein

5.) change in cellular metabolism, function, or development triggered by the receptor-signal complex

6.) Removal of the signal which often terminates the cellular response

1.) Synthesis and release of signaling molecule by signaling cell

includes proteins, small peptides, amino acids, nucleotides, steroids, retinoids, fatty acid derivates, and dissolved acids → extracellular signal molecules

3.) detection of the signal by a specific receptor protein

Two types of receptors

• cell surface receptors → span the lipid bilayer, four main types

  • ion channels

  • G protein-linked → activate a heterotrimeric G proteins

  • linked to an intrinsic enzyme (kinase)

  • linked to an extrinsic enzyme (kinase that binds to the receptor)

ion-channel coupled receptors

change permeabilty of the plasma membrane to selected ons, altering membrane potential and if conditions are right electrical currents

G-protein coupled receptors

activate g proteins which activate or inhibit an enzyme or ion channel in the plasma membrane, initiates intracellular signaling cascade

Enzyme- coupled receptors

act as enzymes or associate with enzymes inside the cell but when stimulated they can activate a wide variety of intracellular signaling pathways

• ligand-binding domains on the outer surface of the plasma membrane

4.) intracellular signaling molecules transfer signals from receptors at the plasma membrane to target protein

three important types of intracellular signaling molecule

• adaptors are proteins that bind to two different proteins linking them together → no enzymatic activity

• 2nd messengers are small molecules that can diffuse into different compartments and also can act to amplify signal

• Switches → signal turns on protein/switch

  • kinases add a phosphate group and phosphatases remove the phosphate group

  • G proteins are active when bound to GTP and inactive when bound to GDP

5.) change in cellular metabolism, function, or development triggered by the receptor-signal complex

-often results in new protein synthesis

6.) Removal of the signal which often terminates the cellular response

-too much signaling can be bad (cancer, inflammation)

-need to be able to regulate strength and duration

Receptor Tyrosine Kinases (RTK)

-class of enzyme-linked receptor proteins

-have a tyrosine kinase domain in the intracellular part of the protein

-ligand binding triggers dimerization of the receptor and activates the tyrosine kinase enzyme portion of the receptor

- when the kinase is activated it cross-phosphorylates the other receptor

-often signal the growth or proliferation of target cells

-turning off RTK can occur by down-regulating RTK, dephosphorylation of RTK, Ras hydrolyzing GTP to GDP and the dephosphorylation of phosphatidylinositol

Ras/MapK pathway

-Ras is a small G protein that is active when bound to GTP and inactive when bound to GDP

-RTK binds to Grb2 → Grb2 binds to Ras-GEF Sos (Sos activates Ras by swapping GDP for GTP)

-Ras then activates MAPKKK which phosphorylates and activates MAPKK which phosphorylates MAPK .

-Active MAPK stimulates cell proliferation, promotes cell survival or induces differentiation by regulating protein activity and changes in gene expression

PI3K/AKT Pathways

- IGF1 binds to the IGF1 receptor it binds to IRS which acts as an adaptor

-IRS binds to the Phosphatidyl inositol 3 kinase (PI3K) - DOES NOT phosphorylate a protein instead it phosphorylates phosphatidylinositol (PI) which creates docking sites for AKT and another protein kinase

-Akt is phosphorylated and activated by two protein kinases

• can phosphorylate Bad or activate mTor (active Akt)

  • Bad activates Bcl2 to increase cell survival

  • mTor leads to changes in protein and glucose metabolism and overall survival

G Protein Coupled Receptors (GPCR)

- has 7 transmembrane domains

-largest cell surface receptor family

Heterotrimeric G Proteins

- larger G proteins are larger G proteins made up of 3 subunits → alpha, beta, gamma subunits

-regulated by GDP and GTP binding → exists as a heterotrimer when the alpha subunit is bound to GDP

-ligand binding to the receptor causes a conformational change in the receptor and the Galpha binds. GDP is released and swapped with GTP and is activated.

-Active Galpha binds to another membrane protein protein to further activate signaling

-to turn off signaling → Galpha hydrolyzes GTP to GDP and rebinds Gbeta/gamma

Gs alpha

-adrenaline activates the beta-adrenergic receptor which activates Gsalpha

-Gs alpha binds to and activates adenylyl cyclase which convert ATP to cAMP

-to turn off signaling → cAMP phosphodiesterase converts active cAMP to AMP

cAMP

-second messenger ( small molecules that activate the next part of the pathway)

-binds to and activates kinase PKA

PKA

-has multiple functions

• activate glycogen breakdown and inhibit glycogen synthesis → fast signaling event

• increases transcription of specific genes by activating transcription factor in the nucleus → slow signaling event

Activation of phospholipase C

-Gq alpha which activates phospholipase C which cleaves PIP2 to DAG and IP3

• IP3 activates a release of Ca2+ from the ER

• PKC is activated by binding to DAG and Ca2+

-Phospholipase C activates a number of end results depending on the cell type

Steroid Hormones

-derived from cholesterol → cortisol, estradoil ( estrogen)

-are hydrophobic and can diffuse across the membrane

• when inside the cell they bind to receptors then enter the nucleus to bind to DNA and to turn on transcription of specific genes

Crosstalk

signals from multiple pathways converge on one intracellular mediator and change the outcome of signaling

endocrine signaling

the most public style of cell-cell communication is broadcasting the signal through the whole body by secreting it into the bloodstream or sap (plants) → long range

• ex. hormones

paracrine signaling

signal cells diffuse locally through extracellular fluid → local neighborhood

autocrine signaling

signal to the same cell → self

contact dependent signaling

cells make physical contact through contact with a signal in the plasma membrane of the signal cells and receptor proteins embedded into the plasma membrane → require contact