Key Concepts in Cell Biology and Microscopy

These flashcards cover key concepts from a lecture on microscopy, actin dynamics, cell transport, organelles, and cell signaling. They are designed to assist in understanding essential cell biology theories and mechanisms.

What are the three key types of microscopy mentioned in this module?

Fluorescence microscopy, Confocal microscopy, Electron microscopy (EM).

What is the purpose of Cryo-EM in structural biology?

Cryo-EM allows the visualization of individual atoms in a near-native state, preserving specimens for detailed imaging.

What is the Raleigh equation used for?

The Raleigh equation helps determine the distance between points in microscopy based on wavelength and numerical aperture.

What are the components involved in cellular transport via microtubules?

Kinesins (towards the ER) and Dyneins (towards the exterior) transport molecules along microtubules.

What role does ATP play in actin dynamics?

ATP hydrolysis leads to a treadmilling effect in actin filaments, facilitating polymerization and depolymerization.

What is the significance of G-actin and F-actin in the cytoskeleton?

G-actin is the globular form of actin, whereas F-actin is the filamentous form that forms the cytoskeleton structure.

Explain the roles of these protein in control of actin filaments:

Cofilin, Profilin, CapZ, Tropomodulin, Formin, Gelosin and Thymosin B4

Cofilin - Binds ADP-bound actin and stabilises the filament.

Profilin - binds G acti, allowing addition at the +ve end

CapZ - binds at the +ve end, caps newly formed ends preventing addition, inhibited by PIP

Tropomodulin - binds -ve end, stabilising filaments in RBSs and muscle

Thymosin B4 = binds ATP-G-actin inhibiting addition at either end of the filament = buffer

Gelosin: regulates Ca2+ concentration, it inserts itself in the chain, causing breakage, and creating more -ve ends fro disassembly.

What are the functions of the Arp2/3 and FH1/2 complexes in actin polymerization?

Arp2/3 complex initiates actin filament nucleation and branching.

FH2 dimers bring 2 G-actins together > F1 forms profilin-actin-ATP complex > F2 adds G-actin to the filament.

What is the role of GEFs and GAPs in Rho GTPase signaling?

GEFs activate Rho GTPases by facilitating GDP to GTP exchange, while GAPs stimulate GTP hydrolysis, turning them off.

What is the endosymbiont hypothesis related to cellular organelles? Explain what is meant by the Schnepf theorem.

The endosymbiont hypothesis suggests that organelles like mitochondria and chloroplasts originated from symbiotic bacteria.

Schnepf theroum: membrane separates plasmic from non-plasmic phase, and only one phase fuses with another phase of the same type.

What is the importance of glycolysation in proteins? Explain what is meant by N and O-linked glycolysation and give examples.

Glycosylation enhances protein stability and function, playing roles in cell signaling and recognition, forming a layer of oligosaccharide/polysaccharide chains facing the extracellular environment.

N-linked: ER, link NH2 groups to Ans side chains

O-linked: Colgi, link to Oh groups in Thr/Ser side chains (calnexin retains unfolded proteins in the ER)

Blood groups: Glycosyl transferase adds monosaccharides to O-antigen (GalNac to A, and Galactose to B)

Influenza: Haemagglutanin trimeric spike protein (addition 7 N-linked oligosaccharides) > insertion via lipid anchor

Explain how B barrel proteins are inserted into bacteria, organelles and chloroplasts.

Bacteria: BAM budding model (C. Dong)

• Recruits Omps to outer membrane > BAMa barrel forms a hybrid protein via B segmentation > each strand added nucleates formation of next strand > new Omp buds away from BAM barrel. SurA/sec proteins as chaperones and DegP degradation pathway.

Organelles: Beta barrel switching

• SAM inserts B barrel proteins into EM via lateral gate > open gate formation with SAM506 as a placeholder > complexes of dimeric SAM and SAM50/Mdm10 > mature Tom40 replaces Mdm10.

Chloroplats: TIC/TOC super complex

• TOM complex: recognises signal seq and transports proteins from cytosol into IMS > protein passed into SAM complex for intertion/folding in OM

• Tim23: TIM22 complex in the membrane for insertion. Uses ATP and redox to transport across double membrane.

Explain how actin and myosin filaments regulate muscle contraction.

Relies on the sliding of actin (thin) and myosin (thick) filaments, generating force via Ca2+(dependent on actin-myosin interactions). Power stroke = switch II helix, converter (ATP centre), lever arm (up for recovery, down for power stroke). Regulated by RyR and the troponin-tropomyosin complex.

Shown by CryoEM resolution of crosslinking proteins e.g. MyPB-C.

Explain what is meant by Membrane Contact Sites and give examples.

MCS: 30-50nm, tethered by molecular machines.

MCS (mt/endosome grabbed by ER for division) 2. ER/ Golgi vesicular trafficking, 3. Diffusion communication.

Lysosomes and Peroxisomes: Syt7 binds PIP2 on peroxisomes, allowing exchange of cholesterol (moved to outer membrane by NPC). Dysfunction causes toxic cholesterol accumulation and neurological damage.

ER and Peroxisomes: ACBDs join VAPA/B regulate lipid transport long chain FA, cholesterol precursors and plasmalogens to ER and membrane lipids to peroxisomes.

Mt and ER/: PTPIP51-VAPA/B (exchange Ca and PA for cardolipin synthesis). GSK3B stops contact VAP-PTPIP51/ACBD

Infection via VAP hijacking of viruses: Norovirus NS1/2 binds VAP.

Visuallised via EM resolution imaging and co-immunoprecipitation to determine tethered interactions and measure distances between cells.

Explain what is meant by plasmalogens and how to measure them

Plasmalogens: Produced via cooperation of peroxisomes and ER = ethanolamine containing ether phospholipids

Protect cells from oxidative stress e.g. mylein shelth, preventing neurological damage.

Microscopy (EM) is used to measure trace interactions and abundance of plasmalogens in various cell types and tissues.

Explain the mechanism of COP mediated cellular vesicular trafficking and communication.

COP (coat protein complex) = needed bidirectional trafficking between goli and ER

COPII vesicles: transport cargo ERGIC between Golgi and ER.

> mediate fusion of COPII to bind to TRAPPI (transport porotein particle complex) through SEC23/4.

COPI: Golgi back to ER.

Mutation in COPII machinery (SEC31A) causes nonsense mediated decay = growth retardation and epilepsy.

Give some examples of protein granules and their functions.

P bodies, stress granules, nuclear speckles and nucleoli.

• Membranelles organelles (liquid-liquid phase separation phenomena) to solid amyloid via regulated aggregation process

• Allow filtration, memory storage (year Tup1-cyc8 and mammalian CPEB3 aggregation) and signal amplification (FLASH concentration in histone locus bodies)

PABPC1 = stress granules bound to end of mRNA, froms compartments under stress.

TDP43 = RNA-protein stress granules, cause ALS as are less dynamic and cannot reform to a translational state.

Nuclear speckles: subnuclear structures involved in mRNA processing and storage of splicing factors. Inhibition of mRNA translation via kinase signalling.

How do focal adhesions function in cellular signaling?

Focal adhesions link the actin cytoskeleton to the extracellular matrix via integrins, facilitating communication and signaling.

What do phosphoinositides (PIs) regulate in cellular processes? Give thr structure and subclasses of PIs.

Phosphoinositides regulate cellular signaling, membrane trafficking, membrane identity, act as spatial markers for organelles, and actin cytoskeleton dynamics.

Hydrophobic fatty acid tails anchor into membranes. Hydrophillic inositol head, that can be phosphorlayted at a wide range of positions.

PtdIns, PtsIns3P (eraly endosome), PtdIns4P(Golgi), PtdsIns5P, PtdIns(3,4)P, PtdIns(4,5)P (plasma membrane), PtdIns(3,5)P (late endosome, lysozymes), PtdIns3P.

Explain the roles of PIs in teh PLC pathway, PKC, PI3K pathway, and the nucleus. Explain how to identify PI binding proteins.

PLC: GPCR/RTK activation > PLC hydrolyses PI(4,5)P2 > DAG (activates PKC) and IP3 release Ca from the ER > second messengers drive multiple downstream pathways.

PKC: interacts with RACKs to determine subcellular localisation. PKC mediates phosphorylisation integrates with ERK/ARK signalling cascades to regulate proliferation and differentiation.

PI3K: Phosphorylates PI(4,5)P2 > PIP3 > recruits PH-domain containing proteins (ARK) = transient and tightly controlled (overactivation can cause tumour progression). PTEN (TSG) regulates this.

Nucleus: Nuclear PI(4,5)P2 localises to - Interchromatin granules (splicing speckles), Nucleoli (RNA pol 1), spliceosome components (RNApol II). PPIns interaction modulates PHA finger function in chromatin remodelling complexes and DNS recombination > PIP3 regulates nuclear Ca release, can impact gene expression.

PI binding proteins: Lipid-protein overlays and mass spec and FLAREs > identify PH, FYVE, PX and ENTH domains.

Explain the role of PI(4,5)P2 in control of actin polymerisation, amd give experimental evidence.

• PIP2 binds profilin, shifting between actin-bound and membrane-bound states.

• PIP2 binds Gelosin, preventing severing, releasing gelosin from actin filaments.

• PIP2 binds Actinin, preventing bunding and the ability to form cross-links.

• CDC42 and PIP2 bind WASP/N-WASP, allowing further activation of Arp2/3 for branching of filaments.

• PIP2 inhibits Cofilin, released upon PLC-mediated hydrolysis of the lipid.

Pyrene- actin polymerisation assay: Affect of Cdc42 and PIP2 on WASP

• Cdc42 binds to GBD (PIP2 binds adjacent basic region)

• Dual binding exposes VCA domain > recruits Arp2/3 > nucleated new actin branches = increase fluorescence actin (Lassing and Linberg)

Explain the roles of PI(4,5)P2 in Focal adhesions and Integrin signalling.

Fas link ECM - integrins (bidirectional signalling hubs) - cytoskeleton.

Talin: FERM domain binds integrin tails, and binds PIP2 at F3 domain. Binding disrupts auto inhibition between head and rob = exposes binding sites binding sites for integrin and actin, allowing FA assembly. RAIM and RAP1 regulate Talin through PH domain binding to PIP2.

Vinculin: PIP2 induces vinculin oligomerisation and activation, allowing binding to Talin/actin, reinforcing FA structure.

Rho, Rac, cdc42: regulate actin and FA by PIP5K activation via GTPases. Allows feedback loop - Rac > PIP5K > ^PIP2 > GEFs > sustained Rac activation.

PIP5Ky isoform 1: competes with integrin fro Talin FERM domain = regulation.

Explain the role of PI(4,5)P2 in endocytosis and phagocytosis.

Endocytosis: PIP2 critical form clatharin coated pit formation to capture cargo and bend the membrane. BAR domain recruits synaptojanin to hydrolyse PIP2 and cause vesicle uncoating. Synthesis and degradation cycle of PIP2 drives vesicle formation and maturation.

Phagocytosis: Fc receptor activation. PIP5K generates PIP2 to probe actin polymerisation. Upon intercalisation of pathogen, PIP5K dissociates, PIP2 levels fall and PI3K generates PIP3.

Explain the role of PIP3 in endosomal identity and describe the process of coincidence detection.

PIP3 produced by PIP3K marks endosomal systems. Enzymatic cascades (synaptojanin and PI-4-phosphatases) inteconvert PIs during vesicle maturation. PI species on coat specifies progression to endocytotic pits.

Coincidence detection: allows specificity, simultaneous recognition of lipids and curvature. e.g. EEA1(FYVE + rab5) for endosome fusion, HSR1 (FYVE and ubq) for degradation, and SNXX1 (PX and BAR) for retromer cycling.

Disregulation can cause neuronal diseases such as Parkinson’s and Alzheimers.

Explain what is meant by tight junctions and gap junctions and give some examples of key protein involved.

Multi-protein complexes that link actin cytoskeleton and ECM via focal adhesions and integrins.

GAG/proteoglycans: highly decorated gels, hydration allows rapid diffusion of small molecules through the matrix.

Fibrillin: tensile strength (collagens and elastin), adhesions for cell attachment (fibronectin and laminin).

Fibronectin: interacts with integrins via strong interactions (act as anchors). Two S-S linked glycosylated chains. Play a role in growth, development, wound healing and cancer. 8-10 F3 module allows cell attachment. The RGD loop is the primary interaction site for integrins (PHSRN and RGD same side of molecule to tune interactions).

^ This can be measured using cell attachment and cell spreading assays.

Describe the structure of integrins and the mechanism of the Hybrid Domain Swing.

Structure: a chain (B propellar, thigh and 2 calf regions), B chain (I-like domain, hybrid chain, PSi region, IEG regions and B-Transmembrane domain). Mg2+ binding site in I domain, Ca2+ binding dite in B propeller, S-S bris=dges between thigh and calf, RDG binding site in B chain.

I domains adopt Rossman fold = conmserved DXSXS motif allow binding metal ions.

In vivo crystallography and EM show 3 majopr states: folded over, cross legged and legs apart.

Hybrid domain swing: prescence ligand causes a7 helix to move down, pushes out hybrid domain and pulls legs apart. Head up, legs apart = high affinity.

Explain what is meant by the MIDAS site, and explain the process of inside-out integrin activation.

MIDAS (metal-ion-dependent-adhesion-site) - metal ion coordination pocket in integrin I domain. Binds Mg/Mn/Ca, forming interactions with acidic residues from ligand, anchoring ligands to the integrin head.

Inside-out signalling: triggers αIIbβ3 TM dissociation and reconstruction of B3 = legs apart

• Membrane-bound Talin binds conserved NPLY motif on integrin B3 tail.

• Phosphotyrosine binding site with Talin FERM domain

• Replacement of aIIb D723-R995 b3 interaction with salt bridge D723-K324 Talin.

• Reorients B TMD, allowing increased affinity for extracellular ligands.

Name the key regions in Focal Adhesion site and how they are regulated.

Adaptors and recruiters - Pascillin for further binding

Signalling proteins - FAK fro phosphorylation

• TyrK, FAT and FERM domains control kinase activity, activity, cell spreading, proliferation, apoptosis and migration

Structural Scaffold proteins - Talin/Vinculin have mechanistic roles.

Integrin-FAK-pascillin-Vinculin-Talin-VASP-actin (layers)

FAK autophosphorylation via PI(4,5)P2 and Sac phosphorlation allow downstream signalling.

Explain how platelets use integrins for signalling.

aIIbB3/2 > GPCRs activation GEF1 / RAP1 pathway = formation RAP:GTP- RAIM - Talin complex > binds NXXY/F motif in integrins = outcompetes a/b tail self=interaction of integrins and promotes integrin activation, leading to increased affinity for fibrinogen and other ligands, facilitating platelet aggregation and clot formation.

What are the major integrins involved in cell adhesion?

Integrins are composed of alpha (8) and beta (18) chains that mediate adhesion and signaling between cells and the extracellular matrix. They form heterodimers (24).

What is the relationship between mechanical tension and talin activation?

Mechanical tension causes talin to unfold and activate, which facilitates actin binding and focal adhesion stability.

What are the consequences of mutations in integrin genes?

Mutations in integrin genes can lead to impairments in inflammatory responses, immune function, and developmental defects.

What role do chaperones play in protein folding within cells?

Chaperones assist in the proper folding of proteins and prevent aggregation, ensuring proper cellular function.

How does PIP2 influence actin polymerization?

PIP2 binds various actin-regulating proteins, promoting actin filament dynamics and polymerization.