Cell biology

Actin

if singular then G actin (normally complexed like in F actin)

Actin treadmilling

Actin ages and moves across the fillament and eventually drop off

GTP binding proteins sit in an active state when GTP is bound to them allowing downstream target effector proteins (kinases and formins) that remodel actin

The cycling is through the GEFs and GAPs/GDIs that activate and inhibit respectively

Branched and linear filamments

Branched formed by ARP2/3 - these are created either by Rac (and subsequently the WAVE complex) or Cdc42 (and subsequently the WASP complex)

Formins form the linear - created by Rho

How the Cdc42, Rac and Rho work in tandem on actin during cell migration

Cdc43 creatred finger like filopodia that sense at the leading edge

Rac Pushes the leading edge forward via the lamellipodia

The Rho forms contactile stress fibers in the cell body and rear that squeezes the cell and pulls the trailing edger forward

ARP2/3

actin related protein that is a seven subunit protein that stimulates the nucleating branched actin networks in eukaryotic cells

Mimics actin dimer/trimer providing a stable seed allowing rapid polymerisation to begin. It creates a branched network that mechanically pushes the cell membrane forward and is the engine behind the formation of lamellipodia and filopodia stimulated by the Cdc42 and Rac complexes respectively

NPFs

They are nucleation promoting factors that activate ARP2/3

They bind allowing the Arp2 and Arp 3, that is bound to the actin filament, to shift closer together mimicking a stable actin dimer allowing free actin monomers to attach and form a daughter filament

Pyrene assay

Pyrene labelled G actin increases its fluorescence when it polymerises to F-actin as the pyrene tag becomes buried in a hydrophobic pocket protecting it from water.

The increase in fluorescence indicates the proportion of actin that is polymerised and can be measured on a fluorimeter graphing actin over time

The graph rises and will only plateau when the reaction has reached equilibrium between the actin subunits adding on and falling off of the filament

Formins

Nucleate and elongate long, straight chained actin filaments and the effectors of Rho GTPases

What is FH and what does it do?

formin homology domain (FH)

FH1 for profilin binding and FH2 for actin polymerisation

FH2 forms a ringed dimer structure around the barbed end of the actin filament and the action of Rho and Rac cause the opening of the ring allowing G-actin to enter

FH1 is a flexible unstructured arm rich in proline and acts as a conveyor belt directing profilin-actin complexes to the FH2 ring accelerating filament growth

F-actin crosslinkers

A protein that binds to F-actin and jins actin filaments together.

Bundling proteins align filaments in parallel or tight arrays like microvilli, filopodia and stress fibres

Network-forming proteins link actin filaments at wide angles creating gel like meshes under the plasma membrane (Alpha actin is a prominent example)

Glutamylated microtubules - influence interactions with MAPs

Detyrosinated microtubules - stabilise microtubules

Tubulin

assembles to microtubules which undergo dynamic assembly and dissassembly due to GDP/GTP, posttranslational modification and tubulin binding proteins

Intermediate filamments

Structural stability, mechanical, apart from the lamins they tend to be expressed in a cell-type specific manner - the lack of depolymerising or stabilising drugs has hampered research

What are the drugs that interfere with microtubuli? (CNT)

Colchicine - binds tubulin and inhibits polymerisation

Nocodazole - depolymerises microtubules

Taxol - stabilises microtubules

What are the drugs that interferre with actin filaments? (PCLJ)

Phalloidin - bunds and stabilises F-actin

Cytochalasin - binds to barbed end, blocks polymerisation

Latrunculin - Binds to actin monomers, prevents their polymerisation

Jasplkinolide - Promotes polymerisation, prevents phaloidin binding

The cytoskeletons role in cancer

actin filaments form structures like lamellipodia and invadopodia to push through tissue, degrade the extracellular matrix and power cell movement

microtubules stabilize cell shape, guide directional movement and handle rapid cell divison

Intermediate filaments provide flexicbility allowing cancer to squeeze through tight spaces during metastasis

Cancer cell metastasis

hyperactivation of Rho GTPases and the Arp2/3 cpmplex drive cancer cell metastasis - Rac and Cdc42 hijack the Arp2/3 complex and build invadopodia that concentrate matrix degrading enzymes like MMPs at their tips creating a physicla path through the surrounding tissue

Alpha actin structure and function

Anti parallel homodimer with each subunit having an Actin Binding Domain (ABD), at the N terminus, and a central rod of four spectrin repeats facilitating dimerization of the 2 chains and the Carboxy-terminal

How - Extended spacing leaves room for myosin II to step in between the filaments and generate cellular contraction

Microtubule basics

Subunit is tubulin that comes in an alpha-beta heterodimer that is tightly bound

The dimers stack end to end forming a linear strand called a protofilament that associate to form a hollow tube (13 protofilament circumference)

Structural polarity created by the dimers facing the same direction. The plus end is terminated by beta tubulin and the minus end is by alpha

They switch from phases of high polymerisation and depolymerisation called Dynamic Instability

What is needed to culture cells? FOOD

A culture medium - salt solutions, pH sensor, glutamine

Growth factors - addition of animal serum

Supplements - Antibiotics, insulin

What is needed to culture cells? ACCOM

Plastic vessels - petri dish, culture flask, multiwell plates, microscopy slides

substrates - extracellular matrix, hydrogels, fibrin matrix, biocompatible scaffolds

What are the 4 types of contamination in culture growth?

Moulds

Yeast

Bacteria

Mycoplasma

How do fluorochromes work?

Shine light on the dye of a specific wavelength causes excitation and a photon is emitted, that was absorbed prior, in return to ground state

What is FRET

Fluorescent resonsance energy transfer

• Used to measure distances and interactions between molecules at a nanometer scale

• Energy is transferred without the emission of a photon from an excited donor to a nearby acceptor fluorophore

What is FRAP

Fluorescent recovery after photobleaching

• Used to investigate protein dynamics in cells

Why is apoptosis important?

ESsential during development

50-70 billion cells a day

elimination of infected or malfunctioning cells

Necessary for menstruation and other physiological processes

Development of the immune system

Misregulation results in disease

Necrosis vs apoptosis

Necrosi

• sewvere sudden injury

• Plasma membrane is the main site of damage

• Leakage if intracellular contents

• Inflammatory response

• Necroptosis is programmed necrosis

Apoptosis

• Programmed cell death

• No leakage of intracellular contents therefore no inflammatory response

• Rapid clearance of apoptotic cells

How is apoptosis regulated?

• signals to stay alive (trophic factors) aren’t present so apoptosis is undergone

• Some cells are triggered to undergo apoptosis by specific signals

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Sculpting of digits requiring apoptosis

The hand starts in a flat paddle shape and the apoptosis of cells produces the finger shapes and then the macrophages remove the apoptotic cells

Morphological changes during apoptosis

Externalisation of membrane phosphatidylserine, Blebbing and osmotic gradient maintained by sealing (transglutaminase)

Shrinkage and condensation of chromatin + endonuclease activity cleaving into DNA fragments

PtdSer Externalisation

Phospholipid scramblase - calcium activates PLS

Flippase - ATP is needed to constantly flip PtdSer back to the inner leaflet

After Apoptosis, the flippase isn’t activated and the PtdSer is left on the outer leaflet and is recognisable to the macrophages

After degradation, post engulfment consequences such as anti-inflammatory cytokines are released

Labelling and visualisation

Annexin V binding to externalised PtdSer

• AV normally functions in the cytosol to rapair damaged plasma membranes

  • REcombinant AV can be used externally to detect e PtdSer (labelled with FITC

Visualisation of blebbing

• SEM

Separation of membrane and cytoskeleton

• Immunofluorescent microscopy

Rock activation leads to blebbing

RhoA GTPase activates ROCK1 this phosphorylated the light chain and the actomyosin becomes contractile as a result. This contractility causes the plasma membrane to clump together leading to the characteristic bulging

Nuclear condensation

The lamin that sits behind the envelope is broken down so it no longer surrounds the nucleus.

It loses its support structure and allows permeation

This can be seen by using DNA binding dyes. - Acridine orange presents as ORNAGE in apoptotic cells but GREEN in healthy cells

Nucleosome wrapping

During apoptosis, the exposed bits become visible to caspase-activated DNase

The DNase causes DNA fragmentation into sections with smaller molecular wright that can be seen using gel electrophoresis.

Aside from the Agarose gel flow cytometry can be used. - Flow cytometry analyses DNA content graphing cell count against fluorensce intensity. During apoptosis, there is a greater sub-G1 peak

TUNEL assay

Terminal deoxynucleotidyl transferase (TdT)s dUTP Nick End Labelling

• DNA in apoptotic cells is nicked extensively TdT adds biotinylated nucleotides to the 3’ end of teh nicked DNA and enzymes coupled to the streptavadin bind to the labelled bases generating a coloured reaction product.

Without apoptosis, autoimmune diseases and impaired immune clearance can occur as there is a failure to make antigen receptors or to remove autoreactive immune cells. Cytotoxic T cells kill targets by inducing apoptosis so there would be impaired killing of infected cells

T cells engage target cells and cause them to be destroyed

• Recognition of the nonself antigens and formation of a self-nonself complex

• Perforin then makes holes in the infected cells membrane and enzymes enter

in vitro motility assay

fluorescent lable on filaments with the assay surface coated with motor proteins. You can then observe the action of multiple motors in terms of speed, direction, cargo and regulation

Optical traps

In vivo myosin functions in the presence of stress. A bead is placed on either side of an actin fillament and the myosin attaches causing a movement leading to a displacement in one direction

Bead assay

Bead attached to the kinesin and takes steps of around 8nm that are tracked

Tracks are polar (not in terms of charge but in direction of movement) and motors perform cyclical and asynchronous interactions with the monomers on a track

Processive motors can move for long distances without detaching the track

Non-processive motors can only take one step such as in muscle myosin and if it isn’t detached, then it will act as an inhibitor to the movement of the track

The ATPase cycle of myosin

Myosin II is tightly bound to the actin filament in the absence of ATP

• The rigor state is when myosin is bound to actin without ATP/ a nucleotide and is normally very transient

• The binding of the ATP causes myosin unbinding

• HYdrolysis of ATP causes a conformational change of myosin to the prime state

• Myosin binds to the actin and releases Pi which generates force when coupled with the release of ADP (another force generating step) the overall power stroke is created that leads to the movement of the actin and resetting the cycle back to the rigor state

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Lever arm

The amplitude of the working stroke is dependent on the length of the lever arm which can be seen in the difference between myosin II in muscle (with shorter arms ) and Myosin V in the AFM (with longer arms) having a longer step

Myosin V

Has a hand over hand movement fashion with 2 heads working alternately

Kinesin 1 ATPase cycle and processive movement on th emicrotubule

Kinesin-1 is tightly bound to tubulin in th epresence of ATP

ADP exchanges for ATP in the leading head that throws the lagging head forward.

Cytoplasmic dynein

Rotation of the microtubule binding stalk

The + end is typically at the periphery of the cell and cytoplasmic dynein is responsible for almost all minus end directed transport. K1 is the best characterised for + end movement but K2 and K3 can play a role

Models for bidirectional transport

Competition between motros - as athere are competing activities and numbers, motors define the direction of trave;

Biochemical coupling - Interactions between motors on a cargo co-ordinate their activity

Axonal transport

ensuring that synapses grow in the right direction. This requires biderectional transport incorporating dynein and kinesin

The turnaround zone checkpoints at the tip of the axon, Dynein is activated and kinesin id repressed allowing transport back to the cell body

Trafficking of melanosomes by K1 dynein and myosin V

Melanosomes have a dispersed state where cAMP is decreased and aggregated when cAMP is increased. The cyclic AMP is what can dictate the activation of the kinesins, dyneins and myosins.

Increased cAMP therefore indicates an increase in kinesin activity as the dispersion is seen away from the body and towards the periphery

Myosin II

Has 2 heavy (motor domains) and 2 light chains (regulatory)

Axonems and dynein function

Microtubule structure with 9 sets of microtubules with 2 sets of microtubules in the centre

The filament can not slide due to the array organisation - regularly spaced - and attached by strong crosslinks Dynein activity creates bending instead of sliding and leads to the waving motion of flagella

regulating the track

The myosin II motor in muscle is controlled by troponin, tropomyosin and calcium ions. tropomyosin blocks the actin binding site and troponin complex. troponin C binding calcium leads to a conformational change in the complex leading to the movement of tropomyosin away from the binding site.

Myosin based regulation of muscle contractility

In the OFF state of myosin (interactive head motif) where the blocked head and free head are folded back on the filament preventing ATP binding. This regulates the consumption of ATP “saving energy” at rest

Myosin motors are activated by the force applied on the filament (mechanosensing) or by phosphorylation of the regulatory light chain

Dynein composition

Vesicle with the cargo and the membrane glycoprotein attached by ankyrin are all needed

Dynein by itself is inactive and does not stably associate with dynactin. Binding to the cargo allows the formation of a tri partite complex and only this tripartite complex is capable of processive motility.

Er and golgi

golgi - stacked sheets and flattened cisternae - site for packaging and glycosylation of secretory cargo

ER - interconnecting tubules and flattened sacs - lipid biosynthesis and site of synthesis of transmembrane proteins

COPII coat

Transitions from the coat to a COPII vesicle that facilitates the movement of cargo from the ER to the golgi.

Sar-1 (the GTPase)

Sar1 - GDP interacts with Sar1-GEF after entering the ER through the ER exit sites. This allows the GDP to be swapped for a GTP as it is catalysed by the GEF (Guanine nucleotide exchange factor).

GTP undergoes a comformational change that exposes an amino terminal a,pithatic helix that inserts into the outer leaf;let of the ERs lipid bilayer.

Activated Sar1-GTP recruits the inner coat heterodimer Sec23/24 with Sec24 binding to the export signals on transmembrane cargo proteins

The movement of cargo after entering the golgi

SNARE mediated fusion with target membrane - to the: endosomal network, plasma membrane or the secretory granules

Constitutive vs regulated secretion

The endocytic pathway

How the cell takes in material from the outside - The end point of the pathway is the lysosome

Degradation of proteins in clells

Ubiquitinated and degraded by the proteasome

Transmembrane proteins are ubiquitinated and sorted to lysosomes

Lysosome funvtion

acidic with a variety of enzymes for degradation of biological content.

An ATPase on the lysosome breaks down ATP and pumps hydrogen into the lysosome causing acidity

How is uptake into the endosomal system facilitated

Phagocytosis by large Plasma membrane folds

Pinocytosis - cellular drinking - non-specific uptake of fluid phase by smaller PM folds

Receptor mediated endocytosis - signal mediated uptake of cargo

Calthirin mediated endocytosis

Three legged protein clathrin

Recruit adaptor proteins into bud that are able to recognise carbon and a neck structure is created and the vesicle is hydrolysewd to separate it from the membrane

Incorporating cargo into the vesicle

Adaptor proteins recognise short linear sequences in cargo that need to be internalised and the arms of the adaptor connect the vesicle to the clathrin protein

SCission of vesicle s using Dynamin GTPase

Separation of the neck by GTP hydrolysis either by wringing or extending the neck on the vesicle

Uptake of cholesterol theought eh endosmal network

The low density lipoprotein receptorbinds to an adaptor due to the AP2 bond facilitated by the NPXY sequence. This allows the uptake of the LDL into the vesicle

Statin funvtion

Prevents the de-novo cholesterol synthesis _ increased endocytosis of LDL-R - circulating levels of free LDL drop leading to increased extraction of cholesterol and decrease risk of Artherosclerosis