Lecture 2:

Cell Biology interactive media ”video” or ”interactive”

1Cell biology 2014 (revised 21/1 -14), Note Lecture 2 handout.

Alberts et al5th edition

Chapter 10617-626628-636

Chapter 11651-664

Chapter 12695-699704-710

A lot of reading!Focus on principlesand topics highlighted inthe lecture synopsis

Ester bond

Membranes are primary built from phospholipids

Phosphate

Glycerol

Fatty acidPhosphoglyceride H

ydrophilic headH

ydrophobic tails

The major phospholipid:

VariableFatty acid

Lipid bilayer5 -8 nm thick

Biological membranes are lipid bilayers primary composed of

amphipathic phospholipids

2

Glycerides (acylglycerols): esters formed from glycerol and fatty acids

Packing of amphipathic lipids in water

Amphipathic lipids will spontaneously form structures that eliminate the exposure of hydrophobic parts to water

- Wedge-shaped lipids form micelles in water

- Cylinder-shaped lipids form bilayers, followed by liposome formation

3

H2O is a dipoleRed: negativeBlue: positive

Movement of individual lipids within the bilayer

Flip-flop (rare)

Phospholipids can freely and rapidly(mm/s) diffuse within the monolayer

The lipid bilayer is a two-dimensional fluid Similar viscosity as olive oil

Spontaneous movements between the two monolayers are rare

Rotational and lateralmovement (frequent)

4video 01.2 crawling_amoeba.mov; 13.5 phagocytosis .mov

Fatty acid length affects membrane fluidity

Long aliphatic carbon chains promote van der Waals interactions decreased membrane fluidity

C=O

CH2

CH2

CH2

C=O

CH2

CH2

CH2

C=O

CH2

CH2

CH2

C=O

CH2

CH2

CH2

van der Waals

van der Waals

van der Waals

Strong interactions Low fluidityWeak interactions High fluidity

Long fatty acid tails Short fatty acid tails

5

Fatty acid saturation affects membrane fluidity

Phospholipids containing only saturated fatty acids

Phospholipids containing a unsaturated fatty acid

C=O

CH

CH2

CH2

C=O

CH2

CH2

CH2

CH2

An unsaturated fatty acid has a kink

6

CH2CH2

CH

Unsaturation's results in steric hindrance decreased van der Waals interactions increased membrane fluidity

Effect of lipid composition on membrane fluidity- Membrane thickness

- Membrane fluidity

Shorter fatty acid chains and an increased degree of unsaturation make a thinner and more fluid lipid bilayer

7

- Interactions between fatty acid chains

Anim. 09.1-laser_tweezer; Video 10.1- membrane_fluidity

Lipid rafts - clusters of strongly interacting lipids

The phospholipid sphingomyelin have long saturated fatty acid tails strong van der Waals interactions Formation of a more static lipid environment

< 100 nm

Lipid rafts are micro-domains of phospholipids with low fluidity8

Inner monolayer (facing the cytosol)Outer monolayerPhosphatidylcholine

Phosphatidylethanolamine

Phosphatidylserine

Sphingomyelin

Percentage of membrane lipids50 40 30 20 10 0 10 20 30 40 50

Asymmetry of the plasma membrane

Phosphatidylinositol, important for cell signaling

Lipid raft former

Extracellular space

-- 9- -molecular_models 10.2-lipids.mov

Different types of membrane proteins

IntegralPeripheral

Single-pass a-helix

Multi-pass a-helixb-barrel

Mono-topic protein Associated to

1.

2.

3.

1.2.3.

LipidIntegral proteinGlycolipid

Integral membrane proteins are not tossed into the membrane randomly, but have a specific topology 10

Dynamics of membrane proteins

Original fluid mosaic model(Singer& Nicolson 1972)

Lipid micro-domain(Simons & Ikonen 1997)

~20 % of the plasma membrane

Lipid raft

11

Rapid movement of proteinswithin the lipid bilayer

Membrane permeability of different molecules

O2CO2

H2O Ethanol

• Small uncharged polar molecules

• Hydrophobic molecules

Benzene

Na+

• Charged

molecules

Ions

N C

H

R

CO

O

H

H

H+

-

Amino acids

Cl-

• Large uncharged polar molecules

Glucose

12

H+

Channel proteins

Creates a hydrophilic channel through the lipid bilayer that isselective for a particular solute

Two types of transmembrane transport proteins Carrier Proteins

Binds a “passenger” at one side of membrane and deliver it to the other side

From above

13

Ion channels

Ion

Ion

Ion

OpenClosed

Ion A Ion B

Ion A

• Most channel proteins are involved in ion transport over the membrane and are therefore called ion channels

• Ion channels are regulated and ion specific

14

Mechanisms behind membrane transport

Simple diffusion

Facilitatedspecific

diffusion Activetransport

Energy independent(down-hill)

Energy dependent(up-hill)

15

Con

cent

ratio

n gr

adie

nt

Different types of active membrane transportTransport of molecules against a concentration gradient requires energy. Cells uses two distinct strategies.

ATP-driven pumps Coupled transporters(symporters)

“Up-hill” transport of molecule coupled to “down-hill” transport of molecule . The “down-hill” gradient depends on a ATP-driven pump

“Up-hill” transport coupled directly to hydrolysis of ATP

PATP ADP +

16

Example of active transport - Na+/K+ pump

Na+

K+

Na+Na+

P

ATP ADP

Na+Na+Na+

P

Na+ 145 mM

5 mM

K+

Na+ 10 mM

140 mM

PK+K+

K+K+

1 cycle 10 milliseconds

1. 2.

3. 4.

Anim. 11.2-carrier_proteins , Anim. 11.1-Na_K_pump 17

Using concentration gradients of Na+ and K+

K+

K+ Na+

Na+

Na+

Na+

Na+

Na+Na+

K+K+

Active transport of Na+ and K+ creates concentration gradients

The Na+ gradient provides the energy for “up-hill transport”

Glucose

1.

2.

3.

GlucoseGlucose

Glucose

Coupled transport of sucrose into the cytosol

1.

2.

3.

The ATP driving the Na+/K+ pump is the energy source for concentrating sugars and amino acids within cells

18

Example of trans-cellular transport by a symporter

Glucose

Na+

Na+Na+

Glucose

Glucose

GlucoseGlucose

1.

2.

1.

2. Active transport: Na+ driven glucose symport (“cotransporter”) 3.

Na+ Na+

Na+

Passive transport: facilitated“specific” diffusion of glucose to blood

3.

Na+/K+ pump establish Na+

gradient

Na+Na+

Na+

Glucose

Intestinal lumen

Glucose

Blood vessels

K+

K+

K+K+

K+

ATP

Anim. 11.3-glucose_uptake 19

Compartment Main functionCytosol Protein synthesis, metabolism

Nucleus DNA & RNA synthesis

Endoplasmic Lipid synthesis, synthesis of proteins that reticulum (ER) enters the secretory pathway

Golgi Sorting and packaging

for delivery to cell

Lysosome Protein degradation

Mitochondrion ATP production

surface or lysosome

20

Compartments/organelles of eukaryotic cells

The nucleus – the instruction book of the cell

Nuclear

pore

1.

2.3.

rRNA +proteins

1.

2.

3.

DNA replication

Transcription mRNA, rRNA and tRNA

Ribosome subunit assembly

3-10 mm

Nuclear processes:

21

One reason for a nucleus in eukaryotes

Transcription

Translation

mRNA processingTranscription

Translation

Prokaryote Eukaryote

In eukaryotes mRNA has to be processed prior to initiation oftranslation, which requires spatial separation of transcriptionand translation (Note cloning of an ORF cDNA synthesis) 22

Transport in and out of the nucleus

Nuclear

pore

Nuclear

porerRNA

mRNA

tRNA

Protein synthesisin the cytosol

DNA replication

1.

2.

1. Transcription

2.

23

The nuclear pore complex (NPC)

Inner nuclear membrane

Outer nuclear membrane

120 nm

Annular subunit; the gatekeeper

Proteins less than 60 kDa can diffuse ”freely” between cytosol and nucleus

A typical cell contains 3000-4000 nuclear pore complexes

24

Nuclear import of proteins (>60kD)

NLSN C

NLSN C

NLSN C

Nuclear Localization Sequence (NLS) = sequence in a protein that mediates nuclear uptake

Could be localized anywhere in the protein

NN CL S

N L SN C

Even distant apart in the primary structureof the protein

Which becomesadjacent in the folded protein 25

The process of facilitated nuclear protein import

Nuclear import receptor (importin)NLS

NLS

NLS

1.

2. 1.

2.

3.

3.

NLS4.4.

Association of target protein and nuclear import receptorin the cytosol

Binding to the nuclear porecomplex mediated by the nuclear import receptor

”Walking” through the gate-keepers of the pore

Dissociation of target protein and nuclear import receptor inside the nucleus

26

The nuclear import cycleCytosol Nucleus

ImportinNLS

ImportinNLS

ImportinGTP

Ran

ImportinGTP

Ran

GTPRan

NLS

Importin RanGDP

Importin

NLS1.

2.

3.

4.Ran

GDP +Pi

<60 kDa

27

The driving forces behind nuclear import

Cytosol NucleusImportin

NLS

ImportinGTP

Ran

GTPRan

NLS

Importin

NLS

ImportinNLS

ImportinGTP

Ran

GTP

GDPEnergy cost!

RanGDP

RanGDP

<60 kDa

28Video 02.3-brownian_motion.mov

G protein

Directionality in nuclear import – the Ran cycle

Cytosol Nucleus

GTPRan

GTPRan

RanGDP

RanGDP

Ran-GAP Ran-GEF

GTPG protein

GDP

GTPase Activating Protein (GAP)

Guanine-nucleotide Exchange Factor (GEF)

Pi

<<GDP GTP

29

Nuclear export

NLS NES

NES

Nuclear export of proteins is mediated by an intrinsic Nuclear Export Signal (NES). Proteins with NES include:

Small protein that shouldnot be nuclear

Protein that shuttle betweencytosol and nucleus

Export of mRNA is dependent on successful splicing

N SE Proteins responsible for splicing

N SE Spliced mRNA ready for nuclear export

Splicing; removal of introns from mRNA

30

Video 12.2-nuclear_import.mov