how are cells studied? microscopy genetics biochemistry molecular biology

How are cells studied?MicroscopyGeneticsBiochemistryMolecular Biology

Light microscopy allows examination of cell morphology

Cells are highly diverse

A protozoan (Didinium) eating another

Bars 10 µm

amoeba

ciliates

euglenoid dinoflagellate

heliozoan

B cellsT cells

Cell shape is determined by a cell wall, or by the cytoskeleton

Structure of Biological Membranes

Cell compartmentalization is achieved by the useof membranes, which are composed of phospholipid bilayers.

Membranes make life on Earth possible, but they also present a great problem, as they impose barriers to diffusion and intracellular

transport

Biological membranes- (e.g. the plasma membrane)-

1. fluidity

3. function

The membrane encapsulates cellular components and maintains an equal solute concentration between the inside and the outside of the cell.A biological membranes’ main function is to segregate chemicals.

Outside

Inside

35-50 Å

2. morphology

Membranes impose barriers to diffusion

Chemical nature of phospholipids-Phosphatidylcholine

Phospholipids are amphiphilic molecules-Hydrophilic and hydrophobic molecules interact differently with water …

Lipids assemble spontaneously into sheets, liposomes and micelles-

A lipid’s chemistry determines its geometric shape(e.g. cones, cylinder, etc.)

Lipids self-associate without covalent bonding; their tails cooperate to exclude water

Different kinds of phospholipids-

* Note their asymmetric distribution in the two membrane leaflets

General function of biological membranes as semi permeable

barriers

Membrane permeability

Membranes function as selective chemical barriers-

The water channel-

Discovery of these water channels led to a Nobel Prize in Chemistry in 1993 to Dr. Peter Agre

Intracellular membranes serve as physical barriers that allow compartmentalization-

Membranes everywhere…

The fluid mosaic model of membrane composition &Topology of membrane associated proteins

Biomembrane composition (a mosaic)-

Proteins are embedded on membranes via hydrophobic surfaces-

Structure of a beta barrel

Hydropathy plot

Hydrophobic tails Transmembrane domains Structure of an alpha helixusually 20 amino acids long

Glycolipid anchor

Fatty acid anchor

Module #1:

A. Cell morphology and organelle compartmentalization B. Membrane structure and function

C. Cellular fractionation and protein topology

Biol110L-Cell Biology Lab-Spring 2011

Budding yeast (Saccharomyces cerevisiae) is a model eukaryotic cell

Our experimental organism of choice

A. Cell morphology and organelle compartmentalization

Lipophilic dyes can be used to visualize membranes-

DiOC6 (low concentration)mitochondria

DiOC6 (high concentration)Mitochondria, ER, etc

Fluorescence microscopy using GFP

Green fluorescent protein (GFP)

Useful when you want to find out the location of a particular protein in cells, to a radius of ~200 nm of its locale

You need to make a gene fusion between the genes encoding GFP and your protein of interest

Cells are not fixed prior to visualization of cells under the microscope; therefore, the technique is used when you want to visualize a protein (a fusion protein) in ‘real time’

Cell peripheryYLR413W (n/a) YEL063 (Can1p) YMR058W (Fet3p)YLR332W (Mid2p)

A collection of yeast strains, each carrying a single GFP tagged protein…

Access the database at- http://yeastgfp.yeastgenome.org/

Access the S. cerevisiae database at- http://www.yeastgenome.org/

for information on each protein

MitochondriaYER080W (Fmp29p) YOR356W (n/a) YGL068W (n/a)

Nuclear peripheryYML031W (Ndc1) YML075C (Hmg1) YOR046C (Dbp5)

Spindle pole

YDR320C (Dad4p) YGL061C (Duo1p)

Nucleoplasm

YER156c YGL097w (Prp20p)

Nucleolus

Cis-Golgi

Yol077c (Brx1p) Ygl078c (Dbp3p)

Yfr051c (Ret2p) Ynl287 (Sec21p)

Vacuole

Cytosol

Ydl185w (Tfp1p) Yor332w (Vma4p)

Ymr235c (Rna1p) Yll024c (Ssa2p)

To expose the yeast plasma membrane for analysis and to weaken the cells in preparation for cell

fractionation, we must first remove the tough yeast cell wall

B. Membrane structure and function

Yeast cell wall composition

The cell wall can be removed with lyticase: a beta 1,3 glucanase(originally obtained from the gut of snails)

C. Cellular fractionation and protein topology in cells

If you want to fractionate cells to isolate an organelle or to determine the cellular distribution* of a protein, use differential velocity sedimentation

Differential velocity sedimentation resolves particles based on size

Low speed pelletLSP

Medium speed pelletMSP

High speed pelletHSP ---> ribosomes, large macromolecules

(3,000 x g)

(15,000 x g)

(100,000 x g)

Low speed supernatantLSS

Medium speed supernatantMSS

High speed supernatantHSS ---> small soluble proteins & molecules

Different types of membrane proteins-

Term used for each protein in this intracellular membrane compartment:

#1: lumenal soluble protein#2: lumenal peripheral membrane protein#3: transmembrane or integral membrane protein (single pass or multi-pass)#4: cytosolic monotopic-integral membrane protein#5: cytosolic peripheral membrane protein#6: cytosolic calcium-dependent peripheral membrane protein#7: cytosolic peripheral membrane protein#8: cytosolic lipid-anchored peripheral membrane protein

Detergents solubilize membranes by dispersing their phospholipids

Detergents

Triton X-100 (a non-ionic detergent) dissolves membranes and solubilizes membrane proteins without affecting their structure/ function.

SDS (an ionic detergent) dissolves membranes and denatures protein structure.

Membrane solubilization with Triton X-100

+

+

Characterization of protein topology on biomembranes-

Subject membranes to centrifugation, which separates soluble (S) from insoluble(or membrane bound or membrane enclosed) material (P).

Analysis of the protein composition of a solution by SDS-PAGE(polyacrylamide gel electrophoresis)-

Used to look at the protein composition of a biological sample. Stain with Coomassie for visualization in the gel

Perform western blot to identify one protein amidst many

Example:Samples from chromatographic column fractions are analyzed during purification of a protein

If you want to visualize a single known protein within a collection of proteins…use Western blotting with specific antibodies-

Transfer proteins from an SDS-PAGE gel to nitrocellulose or PVDF membranes (using electrophoresis), then blot as shown below….

Cell osmolarity- solute concentration

macromolecules organic molecules ions

Cellular mechanisms for dealing with osmolarity issues-

Active ion pumpsCell wall

and turgor pressurein plants

Water extrusion