stupid MCB

Light Microscopy - Bright Field

Phase-contrast and DIC microscopy can be used in time lapse microscopy→ allowing the observer to study cell movement

Magnification - 0.2um

Light Microscopy - Fluroescence

localising molecules within a cell by light microscopy is fluorescent staining of cells and observation by fluorescence microscopy

Magnification - 0.2um

Light Microscopy - Intracellular ion

concentrations within live cells can be determined with ion-sensitive Fluorescent dyes

-         Fluorescent dyes (e.g.. Snarf-1) that are sensitive to H+ concentrations can be used similarly to monitor cytosolic pH of live cells.

Light Microscopy - double label fluorescence

microscopy can visualise the relative distributions of two proteins. This technique involves using two different fluorescent dyes to label specific proteins, allowing the observation of their co-localization and interactions within the cell.

light microscope tagging with fluroescent proteins

allow visualisation of specific proteins in live cells

-         Coding sequence of Green fluorescent protein (GFP) is fused to the coding sequence of a protein of interest–protein of interest is covalently linked to GFP. visualise GFP and therefore protein.

Confocal microscopy transmission (TEM)

1.       Take significant time to scan each focal place → if a very dynamic process is being imaged - the microscope may not be able to collect images fast enough to follow dynamics 2.       It illuminates each spot with intense laser- light → can bleach the fluorochrome being imaged and damage live cells by phototoxicity, thereby limiting the number of images that can be collected

Magnification - 5 um

Scanning Electron Microscope

Electron dense

Views surfaces of specimens that are not sectioned

Magnification - 5um

Difference between indirect and direct immunofluorescence

	Direct	Indirect
Time	Faster	Extra steps - slower
Cost	Conjugated primary is more expensive	Secondary antibody relatively inexpensive
Sens.	Weaker	Multiple secondaries bind to primary

Advantages/ Disadvantages of Direct and Indirect Immunfluoresence

	Direct IF	Indirect IF
Differences	Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore.	Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.
Advantages	Faster time, less complicated.	Greater sensitivity than direct immunofluorescence. Cheaper.
Disadvantages	Lower signal, higher cost, less flexibility, difficulties with labelling.

Do you know the principles of FACS sorting for isolating cells from tissues?

Flow cytometry: fluorescence activated cell sorter (FACS) is used to isolate specific cell types.

Requires fluorescent tag for desired cell.

Processes up to 10 million cells/h

Provides cells for subsequent culturing and propagation.

Can you explain how we can isolate subcellular organelles from tissue or cell cultures? How do we prove that we've been successful in the isolation?

FACS: isolate specific cell types

Subcellular fraction: the study of individual compartments.

DIFFERENTIAL CENTRIFUGATION:

-         Different organelles are separated at different speeds based on their size and density. Nuclei at lower speeds, mitochondria at medium speeds, ribosomal subunits at higher speeds as well as cytosol.

DENSITY GRADIENT CENTRIFUGATION:

-         Organelles of similar mass are separated by centrifugation of a gradient solution according to their equilibrium density.

Organelle purity assessment:

1.      Marker enzymes: measure activities of enzymes that are only found in a particular organelle.

Organelle and marker:

Mitochondrion - Cyt c oxidase, peroxisome - catalase, nucleus - RNA polymerase

2.      EM: to visualise the morphology of specific organelles.

3.      TEM assesses organelle purity.

CELL TO CELL ADHESION:

Occurs through specialised membrane proteins called cell adhesion molecules (CAMS).

CELL TO MATRIX ADHESION:

Occurs through adhesion receptors binding to components of the extracellular matrix (ECM)

Types of Junctions

Junctions	Adhesion type	CAMS of adhesion receptors	Cytoskeletal attachments	Intracellular adapters	Functions
Anchoring Junctions: 1 - Adherens	Cell to cell	cadherins	Actin filaments	Catenins, vinculin	Shape, tension, signalling, force transmission
2 - Desmosomes	Cell to cell	Desmosomal cadherins	Intermediate filaments	Plakoglobin, plakophilin, desmoplakin	Strength, durability, signalling
3 - hemidesmosomes	cell-matrix	Integrins	Intermediate filaments	Plectin, dystorin/BPAG	Shape, rigidity, signalling
4 - focal, fibrillar + 3D adhesions	cell-matrix	Integrins	Actin filaments	Talin, kindlin, paxillin, vinculin kinase	Shape, signalling, force transmission, cell movement

How do cells adhere to the extracellular matrix?

Cell to matrix - Adhesion molecules - often referred to as adhesion receptors , reason why we call these receptors is because there's often ligands or things that bind to the receptors in the extracellular matrix

The cells adhesion molecules that will bind the cells together they are attached to some kind of linker molecule - and that’s usually attached to some type of cytoskeletal feature

Can you list and discuss the different families of Cell Adhesion Molecules (CAMs) and Cell Adhesion Receptors? What is important about their molecular structure? Have you considered the experiments discussed in the lectures that help us understand how these molecules have their function?

Four major families of cell-adhesion molecules and adhesion receptors that exist

1.      Cadherins

2.      ig superfamily

3.      Integrins

4.      Selectin

CAMS promote cell-cell adhesion, which involves both cis and trans binding interactions which are mutually reinforcing.

Include selectins: special example of CAMS that promote leukocyte-endothelial wall adhesion.

Calcium dependent CAMS: integrins, cadherins, selectins.

Calcium independent CAMS: Ig-superfamily.

Can you compare and contrast the different types of cell-cell junctions in terms of: 1) the structure and function of the junction; 2) the types of molecules involved; 3) the important adaptor molecules; 4) the cytoskeletal attachments in each case.

Junction	Adhesion type	CAMS/Adhesion receptor	Cytoskeletal attachments	Intracellular adapters	functions
Tight junctions	cell-cell	Occludin, claudins, JAMs	Actin filaments	ZO-1,2,3, PAR3, cingulin	Controlling solute flow, signalling + polarity
Gap junctions	cell-cell	Connexins, innexins, pannexins	Via adapters to other junctions	ZO-1,2,3	Communication, small-molecule transport between cells
Plasmodesmata	cell-cell	Undefined	Actin filaments	NETIA	Communication, molecule transport between cells.

Cell-ECM adhesion:Hemidesmosomes

-          Connects 2 cells together → interacts with the intermediate filament via adapter molecules

The actual cell adhesion receptor molecule involved = Integrin → made up of Alpha and Beta subunitis (both membrane bound units) they attach to different adapter molecules really critically

Epidermolysis

Integrins link to the cytosolic intermediate filament and basal lamina via laminin

Focal adhesions:

-         Attach cells to EXM

-         Involve integrin molecules

-         In a focal adhesion rather than the intermediate filament seen in hemidesmosomes , Actin filament are the cytoskeletal feature of the cell that’s attaching to the ECM that are the ultimate attachment to the cell via a whole range of different proteins

Important protein adapters important for the focal adhesion to operate

-         Talin

-         Vinculin

-         Alpha actinin

-         Paxillin

-         Focal adhesion kinase

-         SRC

These focal adhesions are attached to the ECM via a different protein by  protein called Fibronectin 

-         These focal are non epithelial cells

COMPONENTS OF THE EXTRACELLULAR MATRIX:

COMPONENTS OF THE EXTRACELLULAR MATRIX:

Major ECM components:

Cells (fibroblasts, mast cells, macrophages, capillary cells), water, lipids, ions, proteins (listed below)

Proteins of ECM:

1.      Insoluble (structural): collagen, elastin

2.      Attract water: proteoglycans

3.      Soluble (multi-adhesive matrix proteins): laminin, fibronectin

Protein	Function
Collagen	Provides structural support to ECM. Insoluble.
Elastin	Insoluble, structural.
Proteoglycans	Heavily glycosylated, has covalently linked polysaccharide chains called glycosaminoglycans (GAGs), negatively charged, bind water.
Laminin	Multi-adhesive protein, forms network. Integrins react with laminin.
fibronectin	Integrins mediate cell-ECM adhesions via fibronectin. Adhesive protein.

Basal lamina (epithelial cells)  

the foundation for assembling cells into tissues. A sheet-like network of ECM components. 60-120nm thick and synthesised by the cells that rest on it. Associates with plasma membrane of cells above and thicker collagen fibres below.

Comprised of four ubiquitous protein components:

1.      Laminins: multi-adhesive protein, forms network

2.      Perlecan: proteoglycan, crosslinker

3.      Nidogen (entactin): crosslinker

4.      Type IV collagen: trimeric molecules, forms network

Can you explain how integrins bind to fibronectin? Do you understand the experiments discussed in lectures that help with this understanding?

Integrins are transmembrane receptors that assist in cell-cell and cell-matrix adhesion. Integrins mediate cell-ECM adhesions via fibronectin. Integrin binds to fibronectin via the RDG motif.

 

Can you discuss the steps in leukocyte extravasation?

-         White blood cell wanting to exit a blood vessel wall to reach a site of infection or inflammation

 

We have -

-         Endothelial cells- Blood vessels

-         Leukocyte

-         ICAMs

-         In the white blood cell we have cell adhesion receptors (aL and B2 integrin)

-         Specific carbohydrates ligands on the surface of the white blood cell

 

Within the endothelial cell and within the vesicle there are also specific molecules (CAMs) selectin molecules, usually wrapped in a vesicle - not presented to the cell surface unless something happens

 

 

-         Inflammation occurs within the tissue

-         that inflammation activates a factor = PAF - this will lead P-selectin moving out of its vesicle and being presented to the external part of the endothelial cell - these P-selectin will bind a selectin ligand which starts to slow the leukocyte down (slow rolling interaction)

-         This will impact the integrins and activate PAF - which will bind the receptor for for PAF on the white blood cell

 

 

So the binding of PAF to its PAF receptor will activate integrin molecule which will then attach to ICAM - and a strong interaction persists

-         Now you've got integrin molecules (from the white blood cell)  binding to adjacent endothelial cells , two endothelial cells will then open up and the white blood cell will be able to extravasate right into the tissue itself

 

○       Can you summarise the two pathways of protein sorting?

1.      Pathway 1: Proteins contain signal sequence → ribosomes attach to the ER membrane and translocation is co-translation 

2.      Pathway 2: Proteins do not contain signal sequence → ribosomes complete translation in the cytosol and translocation is post-translation

Can you describe the sorting signals which direct post-translational or co-translational proteins to organelles?

Can you explain the translocation process for proteins to enter the nucleus, mitochondria, peroxisomes and endoplasmic reticulum?

PATHWAY 2

	Nucleus	Mitochondria
Protein	Folded	Unfolded
Chaperones required		Hsp70 + Matrix Hsp70
Signal	+++ Either: -         Continuous sequence of +ve a.a. -         Bipartite sequence of +ve a.a. -         2 different distinct parts that fold to be close together	-         Most contain Mitochondrial Targeting Sequences (MTS) -         Different signals to target proteins to different sub-mitochondrial compartments
Cytosolic Receptor	Importin	N/A
Membrane Receptor
Translocon ** protein complex that mediates protein transport across cellular membranes	Nuclear Pore FG Nucleoporins -         Nuclear Pore is composed of 8 structural subunits surrounding a central channel -         FG nucleoporins line to NPC channel and form a hydrophobic gel-like matrix that regulates diffusion/transport	TOM and TIM components -         Both rich in acidic -ve a.a. -         Outer membrane - TOM 20/22 (import receptor), TOM40 (general import pore) -         Intermembrane space - TIM23/17/44
Energy	Ran protein (binds to importin)	ATP (chaperone function), proton motor force (H+ gradient)
Cleavage	No	Yes Path A No Path B
	Peroxisome
Protein	Folded
Chaperones required	-
Signal	PTS1 signal: signal at the extreme C-terminus PTS2: signal near the N-terminus
Cytosolic Receptor	PTS1: Pex5 PTS2: Pex7 dependent, interactions with Pex5
Membrane Receptor
Translocon ** protein complex that mediates protein transport across cellular membranes	PTS1 - Pex13/14 complex -         Pex13 has YG repeats (similar to FG repeats of the FG nucleoporins)
Energy	ATP for Pex1/Pex6
Cleavage	PTS1 - No PTS2 - Yes

IMPORT OF PROTEINS INTO DE NOVO PEROXISOMES

PATHWAY 1

ENDOPLASMIC RETICULUM
Signal sequences

-         16 - 30 amino acids

-         one/more +ve a.as near N terminus

-         6 - 12 hydrophobic residues that bind the signal recognition particle (SRP)

-         The signal peptide is cleaved from pre-cursor protein\

○       Identify the principal components of the secretory pathway.

○       Outline the final destination of proteins leaving the Golgi.

○       Describe the protein modifications that occur in the endoplasmic reticulum.

1.      Proteolytic cleavage

a.      Removal of signal sequence by signal peptidase

2.      Glycosylation

a.      N-linked glycosylation

Dol transfers an oligosaccaride  to a basic a.a. On a synthesising protein, The glucose is removed leaving the mannose and n-acetyl glucosamine moiety.

N-linked glycosylation is blocked by tunicamycin

3.      Disulphide bond formation (+ rearrangement)

-         Catalysed by protein disulphide isomerase (PDI)

4.      Protein folding and oligomerisation

-         Require molecular chaperones and other ER proteins

-         BiP and lectins (calreticulin and calnexin) act as chaperones to prevent incorrect folding

-         PDI enhances correct folding through creating correct disulphide bonds

Unfolded Protein Response (UPR)

-         Induced by the accumulation of unfolded proteins in the ER and tunicamycin

-         Results in the activation of Hac1 (XBP1) transcription factors. Hac1 increases the expression of proteins that promote protein folding.

-         Increased production of pro-folding chaperones

** retained proteins undergo mannose trimming. Man5-6(GlcNac)2 recognised by OS9 → dislocated across the ER membrane to the cytosol for degradation.

-         Ubiquitin ligase adds 4 ubiquitin to the dislocated proteins as they enter the cytosol. These polyubiquinated proteins are degraded by the 26S proteasome. The 26S proteasome is comprised of a 20s proteasome (catalytic core) with 19S proteasome caps

 

What is the difference between anterograde and retrograde transport?

Anterograde transport = towards the plasma membrane whereas retrograde transport = back towards the nucleus/ER

Retrograde targeting signals:

-         ER lumenal proteins: …KDEL-COOH

-         ER membrane proteins: …KKXX-COOH

Golgi processing

-         O-linked glycosylation

-         Carbohydrate chains attached to the hydroxyl (-OH) group of serine and threonine residues

-         1 - 4 linear sugar residues

-         Complex N-linked glycosylation

-         Carbohydrate chains attached to the amide nitrogen of asparagine residues

-         Several longer branches of sugar residues

○       Explain cisternal maturation of the Golgi complex.

○       Cisteral maturation: the mechanism in which forward transport of cargo occurs. It is when the cargo stays still but the resident enzymes are re-distributed via retrograde transport.

 

○       Demonstrate how proteins can be retrieved if mis-sorted.

○       Identify the principal components of the secretory pathway.

○       Outline the final destination of proteins leaving the Golgi.

○       No targeting sequence:

■       Soluble proteins get secreted from the cell

■       Membrane proteins delivered to the plasma membrane via bulk flow (default pathway = constitutive secretion → unregulated)

○       Targeting signal:

■       To the lysosome (M6P signal)

■       Golgi proteins have carboxyterminal “retention” signals (and are recovered by retrograde transport).

○       Describe the protein modifications that occur in the endoplasmic reticulum.

○       Explain cisternal maturation of the Golgi complex.

○       Summarise vesicle transport within the secretory pathway, including pinching off and vesicle fusion.

Types of Vesicles

Type of Vesicle	Where used
Clathrin-coated -         3 heavy + 3 light chains -         Has intrinsic curvature due to bend in heavy chains	-         trans -Golgi to endosome -         Endocytosis (plasma membrane → endosome)
COPI-coated vesicles	Retrograde transport -         Golgi (trans-medial-cis) -         Cis-Golgi to rER
COPII-coated vesicles	Anterograde transport -         ER to cis-Golgi

Vesicle Budding

-         Initiated by recruitment of small GTP-binding protein to patch of “donor” membrane that contains membrane cargo proteins

-         GTP-binding protein regulates coat assembly

-         Coat proteins bind cytoplasmic face of membrane (include adapter proteins which first bind the cytosolic face of membrane proteins)

-         SNARE proteins are involved in membrane fusion

-         Dynamin polymerises around the “neck” of the budding vesicle. Hydrolysis of GTP by Dynamin results in release of the vesicle from the donor membrane

Secretory Pathway

Know the structures, dynamics and functions of the actin based cytoskeletal system

Structures:

Actin has 2 forms: globular (G-actin) & filamentous (F-actin)

F-actin: fast growing barbed end (+), slow growing pointed end (-)

             Formed by 3x G-axtin

             Forms branched networks in lamellipodia and parallel bundles in filopodia and stress fibres

Dynamics:

Actin filaments undo constant assembly and disassembly = treadmilling

Dynamic turnover regulated by actin binding proteins that promote nucleation, capping, severing and crosslinking of filaments.

Functions:

Generates forces for cell motility

Facilitates intracellular transport of vesicles and organelles along actin tracks

Cofilin - Active Binding Protein

Fragments ADP-actin filament regions (-ve pointed end)

Enhances overall depolymerisation by making more filament at the -ve ends

Releases ADP-actin monomers

Severs filaments at the negative end

Generates fragments that will further dissemble

Provides G-actin for assembly at the positive end

Profilin - Active Binding Protein

Promotes polymerisation

Binds to ADP-G-actin - causes opening of the cleft increasing release of ADP and replace with ATP

Increases the local supply of ATP-G-actin

ATP-G-actin-profilin can bind to the +ve end of F-actin - profilin is released upon this interaction

Does not increase treadmilling rate, but ensures all free G-actin has bound to ATP - assisting in the increase of treadmilling

Ensures free G-actin is bound to ATP

Enhances polymerisation (can only bind to this end)

Gelsolin

Caps and severs

Regulated by Ca2+ ion concentration

Binding to Ca2+ = conformational change -> allows to bind to the side of actin filament

Gelsolin inserts itself between the subunits -> breaks the filaments

Caps the (+) end of the break preventing further assembly

Generation of multiple (-) ends from the same original filaments promotes disassembly => complete disassembly

CapZ

Capping protein

Tropomodulin

Capping protein

Arp2/3

Produces branched networks -> lamellipodium/leading edge

Requires a nucleation promoting factor (NPF - such as WASp) and a side of existing actin filament

WASp

NPF required for Arp2/3

Intramolecular interactions keep WASp in an inactive form WASp becomes active when its RBD binds active CDC42 and the basic (B) domain binds to the membrane lipid PI(4,5)P2 - result in exposure of A/C/W domains (???)

Thymosin-B4

Actin monomer sequestering protein

Formin

Long actin filaments -> stress fibres

Produce long actin filament

G-actin monomers

divided into 2 subunits by ATP-binding cleft

-> -ve end: binding cleft exposed (pointed end) vs. +ve end: ATP binding cleft contact neighbouring subunit (barbed end)

-> formation of helical structures = F-actin

Polymerisation/depolymerisation rates

-        ATP-G-actin binds to (+) end -> ATP eventually hydrolyzed to ADP + Pi

-        Conformational change -> filament = asymmetric & responsible for different association/dissociation rates at each end

-        As Pi slowly released from subunits, there are 3 distinct regions in the filament

Actin-nucleating proteins

-        These can rapidly and locally nucleate actin assembly under the control of signal transduction pathways

Regulation of actin assemble

-        Formins

-        FH2 domains form a dimer - binds to 2 actin monomers

-        Rocking back and forth - allows addition of actin monomers

-        FH2 sits at the (+) end - protect it from getting capped

Rho-GTP activates Formin

-        When Rho-binding domain (RBD) binds to the Rho-GTP - this allows the exposure of the FH2 domain

-        F1 will bind to profilin ATP-G-actin -> accelerating assembly

Actin + endocytosis

-        Actin polymerisation powers endocytic vesicles into the cytoplasm

Listeria

-        Intracellular bacterium

-        Intra and inter cellular movement requires actin assemble

-        Actin comets

-        Required for listeria motility

-        ATP-G-actin

-        Arp2/3 complex

-        CapZ

-        Cofilin

Actin-myosin interactions

-        Actin drive mobility through interaction with myosin

-        Myosins can convert the energy released by ATP hydrolysis to mechanical work

-        Myosins - large family of motor proteins that can move along actin filaments

Class I	Single heavy chain Variable number of light chains Move towards (+) end
Class II	Assemble into bipolar filaments containing two heavy chains Only class involved in contractile functions Short neck Two light chains per heavy chain Move towards (+) end
Class V	Motor with two heavy chains Long neck region with 6 light chains each Tail terminates in domains which bind specific organelles Move towards (+) end

Which end of the actin microfilament, the + or - end, has the nucleotide (ATP) binding site exposed?

Negative end has ATP cleft exposed

Positive end = back of molecule

What are the three phases of actin polymerisation?

Nucleation: Very slow. Initial pool of G-actin monomers. Formation of a nucleus comprising 3 G-actin (monomers) - rate limiting step (can be skipped if we have a nuclei)

Elongation: Diminishing pool of G-actin monomers. Actin fibrs elongate steadily in the presence of ATP/ADP bound monomers

Steady-state: polymers in ‘steady state’ with equilibrium concentration of G-actin monomers.

What is the ‘steady state’?

Equilibrium of growth -> steady amount of G-actin monomers. Neither increasing not decreasing it’s remaining steady

What is meant by ‘actin treadmilling’?

At steady state -> the (+) end is at a greater concentration than the (-) -> (+) end receiving an addition as the (-) is at a loss

Powered by ATP hydrolysis

Regulated by several actin-binding polymers

Basically, the movement in which the (+) end of the actin gains an ATP-G-actin and extends at the (+) end but loses an ADP-G-actin at the (-) creating a shortening.

What effect would you observe on actin filaments if the G-ATP-actin concentration was increased from 0.3 to 0.7uM?

Concentration above both ends = polymerisation

Concentration below both ends = depolymerisation

Actin filament dynamics can be regulated by actin binding proteins. What proteins:

-        Sequester ATP-G-actin? Thymosin-B4

-        Cap actin? CapZ & Gelsolin (+ end), Tropomodulin (- end)

-        Nucleate F-actin? Formin, Arp2/3

-        Cause actin filaments to branch? NPFs, Arp2/3 (NPFs are involved because Arp2/3 can’t do it by itself - should give both in answer)

-        Fragment actin filaments? Cofilin (can only bind where there’s an ATP -> -ve end) and Gelsolin (can bind anywhere)

-        Cause replacement of ADP with ATP in G-actin monomers? Profilin

-        Caps AND severs actin filaments? Gelsolin

-        Crosslinks actin filaments? Filamin, Fimbrin, Spectrin and a-actinin

How does actin function in endocytosis?

Arp2/3 needs an NPF for endocytosis - uses WASp. Actin will be coupled to the growing endosome and that will generate force to draw the endosome in and deliver where it needs to be - pushing on cell plasma membrane

How does Listeria hijack the actin cytoskeleton:

Listeria uses Arp2/3 to generate new actin sitting at the positive end and pushes along as the actin grows - has it’s own NPF (like WASp) on it’s surface - ActA (Acts as A NPF) - promotes Arp2/3 and assist in pushing the microorganism along.

We also need cofilin to promote severing of the negative end - make more available for growth at the positive end (polymerisation) -> pushes forward into branched actin.

CapZ used to cap any branches forming away from the cell that stops the growth of the branch chain -> ensures only growth that occurs is behind the bacterial cell to help push it into a certain direction.

What domains do myosins have?

Head, neck and tail - head makes contact to microfilament, neck for the movement

How does myosin utilise ATP to generate a powerstroke?

1. Binds ATP, head released from actin

2. Hydrolysis of ATP to ADP + Pi myosin head rotates in ‘cocked’ state

3. Myosin head binds actin filament

4. ‘Power stroke’: release of P and i elastic energy straightens myosin; moves actin filament left

5. ADP released, ATP bound; head released from actin

-        Binding of ATP: disengagement from actin

-        Hydrolysis of ATP: cocking head and binding

-        Release of Pi: power stroke

What is the difference between processive and non-processive myosin movement?

Processivity = constant contact

If processive, one of it’s head is always in contact with the cytoskeletal element

Processive = myosin V - 70% on the cytoskeletal element

Non processive - myosin II - 10% but there are MULTIPLE

Think about them as feet/walking

What is the structure of a sarcomere?

Titin - prevents overstretching and holds thick (myosin) filaments in the centre

Nebulin - determines the length of the thin (actin) filaments

CapZ & Tropomodulin - stabilise actin filament length

How do sarcomeres contract?

Contraction is regulated by calcium

Calcium is pumped in and stored within the sarcoplasmic reticulum

Tropomyosin covers the myosin binding sites on the actin filament - troponin changes shape with calcium -> causes conformational change in tropomyosin that exposes the myosin binding sites

= thin-filament regulation

Inactive state = tropomyosin

Increase in Ca -> troponin conformational change -> strips away tropomyosin -> exposes sites to contract (Ca binds to troponin)

How do smooth muscles contract?

No troponin used here

Calcium binds to calmodulin (CaM) - conformational change

CaM activates myosin regulatory LC (MLC) kinase

MLC kinase phosphorylates the MLC

Myosin unfolds and becomes active

= thick-fialment regulation

What role do focal adhesions play in cell migration?

Provide adhesion and they’re focal like dots or feet

What processes within the cell lead to protrusion of the leading edge?

Lamellipodia and filopodia that projects the membrane forward

Formation of branches actin - NOTE: be clear if microtubules or actin - what type of actin? Branched actin? Lamilopodia or filopodia? Critical for movement

What is the role of the Rho family GTPases in cells migration?

GTPases essential for formation of structures for cell migration - regulated

What kind of assay(s) could we use to measure the role of a given protein in migration?

-        Scratch wound - simple, scrape cells away in middle and the other cells tend to migrate = wound closure and we can measure the rate this is happening

-        If we mutate Rho-GTPases we can remove the ability to migrate together -> we can stain cells to see how actin looks

microfilaments vs microtubules

Microfilaments	Microtubules
-        ATP -        1x structure -        ATP binds at (+), ADP binds at (-) -        Movement: treadmilling -        nucleation	-        GTP -        2x subunits -> alpha and beta -        y-TuRC -        Generation of cytoskeleton

	Myosin V	Kinesin 1 & 2	Dynein
Bind to	Actin microfilaments	microtubules	microtubules
Nucleotide free	bound	Strongly bound (leading head)	Bound Straight linker
ATP bound	Head released	Head bound conformation change in linker - swings forward and docks with head - swings tailing head forward (power stroke)	Head released But when minds to microtubule linker becomes bent
ADP and Pi	Head rotates with respect to the neck “Cocked head” Binds to actin	Pi is quickly released	Pi is quickly released
ADP	Release of Pi causes “power stroke”	Weakly bound (trailing head)	Release of Pi causes power stroke Linker straightens
inactive	Folded - regulated by binding cargo	Folded - regulated by receptor on cargo	Requires dynactin to bind to cargo

Know the structures, dynamics and functions of microtubule based cytoskeletal systems

Structure:

Hollow, cylindrical polymers comprised of alpha/beta-tubulin

Nucleate from MTOCs

Microtubules

-        Roles in mitosis and the motile apparatus of cilia and flagella

-        Involved in transport and organisation of organelles

What are the subunits that construct microtubules? What nucleotides do they bind, and where are these nucleotide-binding sites?

-        Dimer of alpha and beta tubular they bind thyronine nucleotides - predominantly GTP

-        GDP binding clef of B is exposed by GTP of A is licked in

-        Alpha cannot hydrolyse GTP but Beta can hydrolyse GDP

What is the structure of a typical microtubule?

Singlet: 13 protofilaments - alpha and beta tubule dimers. When we get a full wrap around there’s a slight angle where the seam (where they come in contact) found in cytoplasm

Doublet: 13 + 10 - found in cilia, flagella

Triplet: 13 + 10 + 10 - found in basal bodies, centrioles

Name two MTOCs found within the cell. How do they nucleate microtubules?

MTOCs: centrosomes, basal body, spindles pole, y-TuRC

They will have y-TuRC somewhere - this contains y-tubulin that is arranged to act as a template to get nucleation to occur - provides a template for the assembly of a/B tubulin dimers into the protofilament array.

What is the dynamic life of a microtubule determined by?

-        Rate of growth

-        Frequency of catastrophes

-        Rate of depolymerisation

-        Frequency of rescues

Most important thing is the catastrophes and rescues (catastrophe = ram horns, dissociating tubules)

What is the structural difference between a polymerising microtubule and a dissociating one?

GTP cap - most critical thing that determines if we have growth or disassemble or the transition from a catastrophe to rescue

Assembly - stable GTP cap - this determines what happens structurally

What is thought to drive microtubule rescue?

GTP islands introduced during repair of microtubule damage.

As long as we have a GTP cap at some point we can go into rescue by hitting a GTP island

What is microtubule capture?

Using dynamic instability to make contact with a ‘target’, with this contact stabilising the molecule.

Microtubule growing and shrinking until it comes into contact with a target - this will suppress any further catastrophe or growth - often finish temporarily because the microtubule has found what it needs - dynamic instability helps it find its target

How do colchicine and taxol affect microtubules?

-        Colchicine sequesters the tubulin dimers so that microtubules cannot be assembled

-        Taxol stabilises microtubules so that depolymerisation (and therefore search and capture) cannot occur. Taxol treats cells that go through mitosis that shouldn't - this is cancer cells not normal cells

In which direction do a) kinesins and b) dyneins transport their cargo?

a)     All kinesins move towards the + end - except for kinesin 14 (move towards the - end)

b)     Dyneins move towards the - end only

How do each of these motors bind their cargo?

-        Kinesins bind directly

-        Dynein requires dynactin

How does ATP hydrolysis drive movement of each of these molecules along microtubules?

-        How does ATP drive these?

-        Dynein similar to myosin but kinesin very different

-        Domains as functional regions - stalks that make contact it’s not the stalk the moves its the linker - what generates this movement?

-        Myosin = neck region, dynein and kinesins = the linker

What is the general structure of cilia and flagella?

Nexons - interlink doublets of microtubules

How do cilia and flagella bend?

Bending - nexon resisting the sliding activity causing a bend. When dynein is switched off, bending is relieved.

What medicates the increased MT dynamics during mitosis?

XMAP215 suppresses catastrophes induced by kinesin 13 byt XMAP215 is inactivated by phosphorylation during mitosis.

Kinesin 13 is potent - usually suppressed by XMAP215 - they compete for whats happening in the subunit and dimers at positive end

Increased MT dynamics is by inactivating XMAP215 by phosphorylation = reduction activity very specifically during mitosis

Take cells, syncrhonise them to mitosis at same stage and harvest them = low levels of XMAP215 interfase = high level of XMAP215

How do sister chromatids come to be aligned at the metaphase plate?

Microtubule search and capture followed by congression that involves bidirectional movement. Multiple motors contribute to this.

Microtubule search and capture grabs them and then attachment

Backwards and forwards movement until they’re aligned - congressional rate

Whole lot of kinesis that contribute to this activity as well as natural growth and shrinkage of MT themselves

Understand the basic principles of signal of signal transduction

Intracellular signal pathway activated by an extracellular signal molecule. These signal pathways causes have individual signal proteins that result in the activation and regulation of metabolic enzymes, gene expression and cytoskeleton proteins.

Hydrophilic vs Hydrophobic signals

Hydrophobic signals do not require an membrane receptor in order to activate it’s intracellular signal proteins.

-        Move through membrane without a  cytosolic receptor

Hydrophilic signals

Hydrophilic signals  require a membrane receptor that activate more transduction properties and second messenger.

Secondary messengers

Many signals are mediated by secondary messengers, that amplify signal.

Four features of signal transducing system

1. Specificity

   1. When signal molecules binds to their complementary receptor. Non complementary signal will not bind.

2. Amplification

   1. When enzymes activite enzymes, the number of signal affected molecules increase geometrically in an cascade.

3. Desensitization/ Adaptation

   1. Receptor activation motivation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface.

   2. Feedback mechanisms that turns off the receptor.

4. Integration

   1. When two signals have opposite effect on a metabloc characteristic such as the concentration of a second messenger X, or the membrane potential Vm the regulatory outcome results from the integrated input from both receptor.

Two major types of cellular responses

1. Changes in activity or function of specific pre-existing proteins

   1. Proteins activated by a covalent change or conformational change.

2. Changes in amount of specific proteins

   1. Making less or more of something to archive specific goal by modifying transcription factors.

Turning signals on and off

Phosphorylation and dephosphorylation – adding and removal of a phosphate group

Two families:

a)     Serine threoninE

b)     Tyrosine

-        Protein kinase ass on P while protein phosphotase romove the P

-        Regulate with ATP hydrolysis

-        Affects and controls activity of localisation

-        Useful with protein-protein interaction, has the phosphate allows for other protein to dock.

G proteins- binding of GTP or GDP

-        Protein is off when GDP is bound.

-        Protein is on when GTP is bound

-        Dissociation of GDP and binding of GTP.

-        Hydrolysis is Accelarted by GEF protins.