endocytosis & exocytosis

what are the types of exocytosis?

• constitutive - usually immediate

  • mucus, glycoproteins of extracellular matrix, blood proteins

• regulated

  • neurotransmitters, hormones, zymogen granules

what is exocytosis?

the fusion of a secretory vesicle to the membrane of a cell.

what are the current experimental systems in membrane traffic?

• grow in lab under simple conditions

• grow on agar for single cell manipulation

• express asymmetry of form - polarised

• genetics - ready isolation of mutants, e.g. discovery of the “Sec” mutants

  • budding yeast - cheap & easy to grow, can induce random mutations → get clones, isolate & analyse

what are SNAREs?

• protein complexes

• need to know where to go

• complementary vesicle (v-SNARE)

• target (t-SNARE)

what are vectoral properties?

• need for control to prevent random fusion of vesicles since such a chaotic situation would kill the cell.

• needs a system of SNARE proteins - matching pairs on vesicle & target → tells vesicle where to go.

• the cell needs mechanisms to ensure directional flow of the vesicle traffic.

• this required the evolution of special proteins (Rab’s) and the need for NTP hydrolysis that prevents anything other than the correct sequence of events

  • effector proteins - ensure vesicle fuse w the right target

  • NTPs = ATP or GTP

what is endocytosis?

the uptake of material into a cell

• e.g. phagocytosis

how does endocytosis look in membrane traffic?

coated pits → coated vesicles → endosome → lysosome

• transport vesicle from early endosome returns LDL receptors to plasma membrane

what is protein targeting?

• proteins synthesised on the cystolic ribosomes

• proteins synthesised on ER membrane-bound ribosomes

• proteins destined for cell organelles contain targeting (gene coded)

what happens when “good proteins go bad”?

• ubiquitin tag

• passed to proteasome (protein complex)

• degraded

• genetic diseases associated with deficiencies in this pathway

• Ciechsnover, Hershel, Rose - awarded for the discovery of ubiquitin-mediated proteolysis responsible for intracellular protein degradation

describe the proteasome pathway

• ubiquitin tag gets attached to lysine residue on protein (ensure specificity)

• target protein with polyubiquitin chain

• moves into channel

• oligopeptides products of proteolysis are released

what are the features of the cytoskeleton?

• very high protein concentration - around 20%, 0.1%=1mg/ml typical conc for experiments

• fluid properties

  • non-newtonian (can behave like a solid for sudden forces)

  • non-uniform - micro domains exists, resists sudden impacts but “melts” under slow, persistent shear

• diffusion by thermal motion

  • fast - ions, small molecules (ATP, tRNA, majority of mRNA)

  • slow - macromolecules, some mRNA - illustrates need for evolution of specialised intracellular transport systems

what is the impact on cell biology from using electron microscopy?

complex membrane systems

• compartments of different metabolic functions

  • single or double membrane bound organelles

  • membrane traffic

cytoskeleton

• structural function (micro-engineering, microtubules) effector functions

  • integration of compartments (traffic) and cell-cell signals

  • organelle distribution

  • cell motility & division - generate forces (e.g. mitosis)

what is the operational definition of the cytoskeleton?

insoluble cytoplasmic residue left after extraction with non-ionic detergent in matched ionic strength buffer, this residue may be analysed by stereo EM & conventional biochemical techniques

what does the cytoskeleton consist of?

consists of polymeric fibrous & tubular elements

• polymers - dynamic (assembly and disassembly rapid), may be polar

• accessory proteins - structural (force dissipation), motor (force production)

  • binding proteins = specificity

  • movement = e.g. muscle contraction

what are the 3 major classes of filament?

• microtubules (polar)

  • tubulins

• microfilaments (polar)

  • actin

• intermediate filaments (non polar)

  • heterogenous composition, purely structural elements only important for multicellular eukaryotic organisms.

describe the response of cytoskeleton polymers to deformation.

• individual actin filaments - easily deformed and ruptured, cross-linked actin bundles are more rigid

• individual microtubules - very rigid, but do rupture easily

• vimentin - networks easily deformed, but withstand large strains and stresses without rupture

describe the evolution of the cytoskeleton

• serial endosymbiotic theory - organelles, genetic system, cytoskeleton - but small cell size would mean that ancestors could rely on diffusion and not vectoral transport, ancestor proteins oriannly had a different function

  • can explain how mitochondria became part of the cell (own DNA)

• actin-like proteins (MreB) - similar to bacteria, bacillus subtilis (actin-like protein involved in maintaining asymmetric shape of cell)

• tubulin-like proteins - FtsZ (similar to beta-tubulin) associated with the fission ring