Cell Bio techniques

steps of inquiry

approaches taken

the techniques covered:

immunofluorescence, EM,

subcellular fractionation: centrifugation

affinity, gel, ion-exchange chromatography

SDS-PAGE

PM characteristics

permeability

mechanical

PM lipid composition

phospholipids: name & key structure

sphingolipids name & key structure

cholesterol: key structure

PM asymmetry

outer & inner leaflet, compartments,proteins

PC, SM out; PI, PS, PE in

PM fluidity: phase transition

chain length, saturation, cholesterol

measured with FRAP, cell fusion

PM proteins

types: TM, lipid anchored, peripheral

ionic & nonionic detergents

TMD: alpha helix, hydropathy plot

topology: protease protection

Simple diffusion

partition coef. implications

Transporters

uniporter: properties, mechanism

glucose

symporter

antiporter

Pumps

P-class: properties, mechanism

Ca pump

Na/K pump

V-class: properties

ABC: properties

Membrane potential

electrochemical gradient

Ion Channels

ligand, voltage, signal gated

mech. for selectivity

mech. for gating

mech. for inactivation

patch clamp/ expression system

Action Potential

ion movements & their basis

myelination

threshold for firing

Nuclear Import/Export

nuclear structure

transport cargo

nuclear pore

targeting signals: NLS, NES

Ran GTPase

Import/Export receptors

FG platform

Regulation

Nuclear Import Study Questions:

1. In what sense does Ran convey cell position to import and export receptors? In your answer provide the background details that explain how it works.

2. What would happen if wild-type Ran were mutated to a form that can bind GTP but cannot hydrolyze GTP?

3. What experimental steps would you take to define the exact sequence and position of a nuclear import signal in a nuclear protein that you discovered?

4. What steps would you take to identify your protein's import receptor?

5. How is directionality achieved for import into the nucleus? Explain mechanism being sure to identify the driving force and where it comes from.

6. How is selectivity achieved in the gating mechanism at the nuclear pore? Explain mechanism.