lecture 2: cell biology interactive media ”video” or ”interactive” 1 cell biology 2014...
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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 10
617-626628-636
Chapter 11
651-664
Chapter 12
695-699704-710
A lot of reading!Focus on principlesand topics highlighted inthe lecture synopsis
Ester bond
Membranes are primary built from phospholipids
Phosphate
Glycerol
Fatty acid
Phosphoglyceride
Hydrophilic head
Hydrophobic tails
The major phospholipid:
VariableF
atty acidLipid 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
CH2
CH2
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 monolayer
Phosphatidylcholine
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-helix
b-barrel
Mono-topic protein Associated to
1.
2.
3.
1.
2.
3.
Lipid
Integral protein
Glycolipid
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_pump17
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
Glucose
Glucose
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 function
Cytosol 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
pore
rRNA
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
Importin
GTP
Ran
Importin
GTP
Ran
GTP
Ran
NLS
Importin RanGDP
Importin
NLS1.
2.
3.
4.Ran
GDP +Pi
<60 kDa
27
The driving forces behind nuclear import
Cytosol NucleusImportin
NLS
Importin
GTP
Ran
GTP
Ran
NLS
Importin
NLS
ImportinNLS
Importin
GTP
Ran
GTP
GDP
Energy cost!
RanGDP
RanGDP
<60 kDa
28Video 02.3-brownian_motion.mov
G protein
Directionality in nuclear import – the Ran cycle
Cytosol Nucleus
GTP
RanGTP
Ran
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 SESpliced mRNA ready for nuclear export
Splicing; removal of introns from mRNA
30
Video 12.2-nuclear_import.mov