Download Lecture 3 – Membrane potential

Q&A session I
Dr. Sara Batelli
[email protected]
08.11.2016
Lecture 1
Cells compartments
Microscopy
Lecture 2
Membrane composition
Lecture 3
Different types of pumps
Action potentials
Uses of pumps and membrane potential together
Lecture 4
Protein Targeting - Nucleus and ER
Lecture 5
Protein Targeting - Mitochodria and Choroplasts
Lecture 6
Protein Trafficking in the Secretory Pathway
All Lectures – Prokaryotic and Eukaryotic cells
Prokaryotic cells



Much smaller
The genetic material is naked
within the cytoplasm
Ribosomes are the only type of
organelle
Eukaryotic cells



A double membrane-bound nucleus
separates the genetic material from the rest
of the cell
An endomembrane system composed of
different membrane-bound organelles that
transport materials around the cell: the rough
and smooth endoplasmic reticulum, Golgi
apparatus and vesicles
Energy producing organelles: mitochondria
and chloroplasts, involved in metabolism and
energy conversion
All Lectures - Cell compartmentalization in eukaryotic cells
All Lectures – Different cellular compartments
Cellular compartments:
 Nucleus → DNA
 Mitochondria → ATP generation
From where do they
come from?
 Chloroplasts → capture energy from sunlight to make sugars
 ER → lipid synthesis/ protein synthesis, storage of Ca++
 Golgi → protein modification/ targeting
 Endosomes → protein sorting
 Lysosomes → protein digestion
 Peroxisomes → oxidation
They are all enclosed by a membrane but each compartment has specific
enzymes, molecules and transport systems on the surface
All Lectures – Different types of transport
Gated transport
(nucleus)
Transmembrane
transport
(ER, mitochondria,
chloroplasts)
Vesicular transport
(secretory pathway)
Lecture 1 – Microscopy (light and electron microscopy)
Light Microscopy - so called because it employs visible light to detect small objects
Resolution is defined as the ability to distinguish two
very small and closely-spaced objects as separate
entities. Resolution is determined by certain physical
parameters that include the wavelength of light (λ) and
features of the objective lens (NA = numerical aperture)
Rayleigh criterion is the general accepted criterion for
the minimum resolvable detail. Two adjacent objects are
resolved when the first diffraction minimum of one object
coincides with the maximum of the other.
Lecture 1 – Microscopy (light and electron microscopy)
Different Types of light Microscopy:
Bright field
Dark field
Phase contrast
Fluorescence Microscopy
A fluorescence microscope is much the same as a conventional light
microscope with added features to enhance its capabilities.
The conventional microscope uses visible light (400-700 nm) to illuminate; a
fluorescence microscope, on the other hand, uses a much higher intensity light
source which excites a fluorescent species in a sample of interest. This
fluorescent species in turn emits a lower energy light of a longer wavelength
that produces the magnified image. In most cases the sample of interest is
labeled with a fluorescent substance known as a fluorophore and then
illuminated. The illumination light is absorbed by the fluorophores (attached to
the sample) and causes them to emit a longer, lower energy wavelength light.
Lecture 1 – Microscopy (light and electron microscopy)
Which dyes or fluorescent proteins are good for imaging?
1) High extinction coefficient (= “brightness”)
2) High quantum yield of fluorescence (= number of fluorescence photons emitted
per excitation photon absorbed)
3) Little photobleaching (= loss of fluorescence signal over time)
4) Absorption in the far red (limits damage to living cells and shows
low autofluorescence)
Lecture 1 – Microscopy (light and electron microscopy)
Fluorescence Microscopy – Wide-field vs Confocal
A pinhole is in front of
the detector to eliminate
out-of-focus signal
Low resolution in Z
High time resolution
High resolution in Z
Low time resolution
Lecture 1 – Microscopy (light and electron microscopy)
Two Types of Electron Microscopy
1) Transmission Electron Microscopy (TEM)
See details of a cell by sending an electron beam
through very thin slices of a specimen. An image is
formed from the interaction of the electrons transmitted
through the sample. It can be coupled with antibodies to
localize specific proteins
2) Scanning Electron Microscopy (SEM)
See details of the outside of a sample. Once the electron
beam hits the samples, electrons are ejected from the
sample and collected by a detector. Computers are used
to bring them back into a 3D image
Lecture 1 – Microscopy (light and electron microscopy)
Two Types of Electron Microscopy
Lecture 2 – Membrane’s composition
Cellular membranes
 Lipid bilayer
 Different proteins
 The plasma membrane
contains ~ 25% protein,
internal membranes (e.g
ER, Golgi…) contain up to
75% protein
Functions of plasma membrane:
 Protection from the external environment
 Import/export of molecules
 Signal transduction (sensing of external stimuli and activation of internal
pathways)
 Cell motility
Lecture 2 – Membrane’s composition
Lipid bilayer
Membrane lipids are
AMPHIPATHIC
The organization of the
lipid bilayer is
ENERGETICALLY
FAVORABLE
What’s the difference?
Saturated lipids
Mix of saturated and
unsaturated lipids
Lecture 2 – Membrane’s composition
3 different types of membrane lipids
Phospholipids
Sterols
Glycolipids
Is the composition of the two layers the same?
Different in the two layers
ASIMMETRY
Only in the outer layer
Lecture 2 – Membrane’s composition
The lipid bilayer is a fluid
Possible phospholipids movements
 Lateral diffusion, rotation, bend
 Flip/flop between the two layers is rare
Factors that influence the lipid bilayer fluidity:
 Temperature
 Composition of the membrane
- Lengh of hydrocardon tails
- Presence of double bonds
- Amount of Cholesterol
Lecture 2 – Membrane’s composition
Membrane proteins
Majority
Lecture 2 – Membrane’s composition
What is represented here in red?
The cell cortex, is a specialized layer of cytoplasmic protein on the inner face of the
plasma membrane of the cell periphery. It functions as a modulator of plasma membrane
behavior and cell surface properties. In most eukaryotic cells lacking a cell wall, the cortex
is an actin-rich network. The cell cortex is attached to the cell membrane via membraneanchoring proteins and it plays a central role in cell shape control. The protein constituents
of the cortex undergo rapid turnover, making the cortex both mechanically rigid and highly
plastic, two properties essential to its function. In most cases, the cortex is in the range of
100 to 1000 nanometers thick.
Lecture 3 – Different types of pumps
Osmotic concentration, formerly known as osmolarity, is the measure of solute
concentration, defined as the number of osmoles (Osm) of solute per litre (L) of solution
(osmol/L or Osm/L). The osmolarity of a solution is usually expressed as Osm/L
(pronounced "osmolar") and measures the number of osmoles of solute particles per unit
volume of solution. This value allows the measurement of the osmotic pressure of a
solution and the determination of how the solvent will diffuse across a semipermeable
membrane (osmosis) separating two solutions of different osmotic concentration.
Are ion concentrations different or not inside and outside the cell?
How can you keep the different ion concentrations inside and outside?
Lecture 3 – Different types of pumps
Transport across the membrane: different types of transmembrane proteins
Concentration
gradient
Pump
Types of transport
Passive – no energy = down the concentration gradient
Active – energy required = against the concentration gradient
Lecture 3 – Different types of pumps
Transport across the membrane
++++++++++++++++++++++++++++++
Membrane
potential
- - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Pump
Electrochemical gradient = concentration gradient + membrane potential
Lecture 3 – Different types of pumps
An electrochemical gradient is a gradient of electrochemical potential, usually for an ion
that can move across a membrane. The gradient consists of two parts, the chemical
gradient, or difference in solute concentration across a membrane, and the electrical
gradient, or difference in charge across a membrane. When there are unequal
concentrations of an ion across a permeable membrane, the ion will move across the
membrane from the area of higher concentration to the area of lower concentration through
simple diffusion. Ions also carry an electric charge that forms an electric potential across a
membrane. If there is an unequal distribution of charges across the membrane, then the
difference in electric potential generates a force that drives ion diffusion until the charges are
balanced on both sides of the membrane.
Lecture 3 – Different types of pumps
Terminology
1) Types of transport
Passive – no energy = down the concentration gradient
Active – energy required = against the concentration gradient
2) Types of Membrane Transport Proteins
Pump –
active and slow (they transport ions against their electrochemical gradient)
light or ATP driven, conformational changes
P-type Pumps (Na, K, H, Ca) self phosphorylate
ABC transporter – small molecule transport
Carrier – mainly passive
Uniporter – single substrate across membrane
Symporter – transport of two solutes across the membrane
Antiporter – exchange of one solute for another across the membrane
Channel – passive and fast (they transport ions with their electrochemical gradient)
They are ion specific pores that open and close transiently, without
conformational changes
Lecture 3 – Different types of pumps
What is the difference between Carrier and Channel Proteins?
• Solutes diffuse through the pore of channel proteins, whereas carrier proteins bind solutes
on one side of membrane and release it on the other side
• Compared with channel proteins, carrier proteins have very slow transport rates
• Unlike carrier proteins, channel proteins contain a pore, which facilitates the solute
transportation
• Unlike channel proteins, carrier proteins have alternate solute-bound conformations
• Channel proteins are lipoproteins, while carrier proteins are glycoproteins
• Carrier proteins can mediate both active and passive transport, while channel proteins can
mediate only passive transport
• Carrier proteins can transport molecules or ions against the concentration gradient, while
channel protein cannot.
Lecture 3 – Different types of pumps
Na/K Pump
Conformational modification
https://www.youtube.com/watch?v=awz6lIss3hQ
Lecture 3 – Different types of pumps
Carrier Proteins
Example : Glucose Transport from gut lumen into epithelial cell
https://www.youtube.com/watch?v=o_uDwQfbyAE
Lecture 3 – Different types of pumps
Stress Gated Channel (Hair Cells)
In lecture we saw that channels are highly selective to ion which they transport based on size
and charge. They don’t undergo a conformational modification. They open after ligand binding,
membrane potential modification or mechanical stress.
https://www.youtube.com/watch?v=zKuor6wK1uU
Lecture 3 – Membrane potential
Membrane potential (also transmembrane potential or membrane voltage) is
the difference in electric potential between the interior and the exterior of a
biological cell
While this phenomenon is present in all cells, it is especially important in nerve
and muscles cells, because changes in their membrane potentials are used to
transmit information. When a nerve or muscle cell is at "rest", its membrane
potential is called the resting membrane potential. In a typical neuron, this is
about –70 millivolts (mV). The minus sign indicates that the inside of the cell is
negative with respect to the surrounding extracellular fluid. If some event, such
as the opening of a gated ion channel, causes the membrane potential to
become less negative, this is termed depolarization. Conversely, if some factor
causes the membrane potential to become more negative, this is termed
hyperpolarization.
Lecture 3 – Membrane potential
Membrane potentials are used to propagate signals along a nerve cell without
loss of signal over long distances
An action potential occurs when a neuron sends information down an axon,
away from the cell body. The action potential is an explosion of electrical activity
that is created by a depolarizing current. This means that some event causes
the resting potential to move toward 0 mV. When the depolarization reaches
about -55 mV a neuron will fire an action potential. This is the threshold. If the
neuron does not reach this critical threshold level, then no action potential will
fire. Also, when the threshold level is reached, an action potential of a fixed size
will always fire. For any given neuron, the size of the action potential is always
the same. Therefore, the neuron either does not reach the threshold or a full
action potential is fired - this is the "ALL OR NONE" principle.
Lecture 3 – Membrane potential
Generation of an action potential
Na+ channels
inactivation
Positive feedback
Na inactivation = The Na+ channels begin to close, even in
the continued presence of the depolarization. Inactivation
contributes to the repolarization of the action potential.
However, inactivation is not enough by itself to account fully
for the repolarization.
Lecture 3 – Membrane potential
Propagation of an action potential
Due to channel inactivation and the resulting refractory period, the action
potential propagates only in one direction → the membrane cannot generate a
second action potential until the Na+ channels have returned from the
inactivated to the closed state
Lecture 3 – Membrane potential
How to increase the speed
of action potential
propagation?
Lecture 3 – Membrane potential
Along the axon:
The myelin sheath insulates, such that
the action potential jumps from node to node
At the axon terminal:
Neurotransmitter
Voltage-gated Ca++
receptors
channelsproduce
convert the
changes
of
the
electrical
signal
membrane
potential
(action potential) into
according
to the
a chemical
signal
amount
of
(release of
neurotransmitter
neurotransmitters)
released
Lecture 3 – Membrane potential
Synaptic Signaling
https://www.youtube.com/watch?v=py4DZ9WRqDM
Lecture 4 – Protein sorting: nucleus and ER
How are proteins targeted to their specific location
within the cell?

Proteins are targeted to specific compartments by signal sequences that
are recognized by specific receptors

Different types of signal sequences (N/C terminal, internal)

Signal sequences are necessary and sufficient for targeting
Lecture 4 – Protein sorting: nucleus (gated transport)
Nuclear pore complex:
 Big complex
 In general is formed by 8 subunits
(nucleoporins)
 Allows the passage of proteins >
60 kDa (gated transport)
 The big proteins should deform in
order to squeeze through the
channel
• Nuclear localization sequence (NLS) required for nuclear import
• Export: NES = nuclear export sequence)
• Also: NRS = nuclear retention signal
Lecture 4 – Protein sorting: nucleus (gated transport)
Nuclear import receptors (importins)
To initiate nuclear import, most nuclear localization signals must be recognized by nuclear
import receptors, sometimes called importins, most of which are encoded by a family of related
genes. Each family member encodes a receptor protein that can bind and transport the subset
of cargo proteins containing the appropriate nuclear localization signal (direct binding). Nuclear
import receptors do not always bind to nuclear proteins directly. Additional adaptor proteins can
form a bridge between the import receptors and the nuclear localization signals on the proteins
to be transported (indirect binding).
Lecture 4 – Protein sorting: nucleus
 How is the cargo protein released?
 What determines the directionality of transport?
Lecture 4 – Protein sorting: nucleus
Ran (GTPase)
GDP
(Cytoplasm)
GTP
(Nucleus)
Lecture 4 – Protein sorting: nucleus
How the Ran-GTP level is maintained high inside the
nucleus?
Cytoplasm
Ran-GAPs and Ran-GEFs
Nucleus
 Ran-GEF
 Exchange GDP
with GTP
 Ran-GAP
 GTP-hydrolysis
Dissociation of Ran
from the transport
receptor
Dissociation of
protein cargo from
the transport receptor
GAP = GTPase activating protein
GEF = Guanine-nucleotide exchange factor
Lecture 4 – Protein sorting: ER
Protein Translocation in the Endoplasmic Reticulum
https://www.youtube.com/watch?v=4qf1BSXn_tk
Lecture 4 – Protein sorting: ER
 Targeting to the ER occurs co-and post-translationally by the
conserved Sec pathway
SRP (= signal recognition
particle) binding causes
translational slow-down. It
binds to SRP receptor on
ER membrane). They are
both GTPases
 Only proteins carrying a ER-signal sequence are targeted to the
ER membrane; translocation occurs via the Sec 61 complex
 Sec 61 can open laterally and vertically to insert membrane proteins or
translocate soluble proteins
 Start-transfer and stop-transfer sequences serve as
insertion/translocation signals
Lecture 4 – Protein sorting: ER
What is the function of the protein BiP?
Binding immunoglobulin protein (BiP) is located in the lumen of the ER, binds newly synthesized
proteins as they are traslocated into the ER, and maintains them in a state competent for subsequent
folding and oligomerization. BiP is also an essential component of the traslocation machinery and plays a
role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the
proteosome.
Lecture 5 – Mitochondria and Chloroplasts
2 membranes = 2 compartments
3 membranes = 3 compartments
specialized on ATP-Synthesis using energy
derived from electron transport and oxidative
phosphorylation
specialized on ATP-Synthesis
derived from photosynthesis
Similarities:
- contain DNA and ribosomes + other components required for protein synthesis
- most of the proteins are encoded in the cell nucleus
- most proteins have to be imported from the cytosol
Lecture 5 – Mitochondria and Chloroplasts
Protein Import
https://www.youtube.com/watch?v=FhJemzfF7rc
Lecture 5 – Mitochondria and Chloroplasts
Translocation into mitochondria:
1)
Generally, N-terminal signal sequence
2)
Different receptors and channels act to translocate proteins
- TOM and SAM complexes translocate proteins into the outer mitochondrial membrane
- TIM23 complex transports soluble proteins into the matrix and helps to insert proteins into
the inner membrane
- TIM22 mediates insertion in the inner membrane
- OXA complex mediates insertion in the inner membrane of proteins synthesized by
mitochondria
- Hsp70: the cytosolic Hsp70 binds to unfolded proteins to avoid aggregation; the
mitochondrial Hsp70 acts as a motor to pull the proteins into the matrix space. They both
require ATP hydrolysis to be released
Energy sources (ATP hydrolysis and membrane potential)
TIM23
Lecture 5 – Mitochondria and Chloroplasts
Protein transport into Mitochondria vs Chloroplast
Mitochondria
Chloroplasts
-
They use membrane potential
and ATP as energy sources
-
They use GTP and ATP as
energy sources for translocation
-
N-terminal signal sequence
-
They use H+ gradient
(membrane potential) for
translocation into the thylakoid
-
For translocation into the
thylakoid, they have 2 N-terminal
signal sequences
Lecture 6 – The secretory pathway: vesicular transport
Does vesicular transport occur spontaneously?
How can you visualized a protein that enters the secretory pathway?
Lecture 6 – The secretory pathway: vesicular transport
(PM)
ER (lipids and proteins synthesis):
Disulfide Bonds – stabilizes protein
Glycosylation – protects against
degradation and aggregation, helps
with correct folding and is involved
in cell-cell recognition and cell
signaling
Quality control – misfolded proteins
are recognized by chaperones
Golgi:
Protein sorting and
distribution center
Protein modification
Glycosylation processing
Final Destinations:
Lysomes – degradation
Vesicle – bind to PM to
secrete or to deliver
proteins to the PM
Can also bind to
endosomes
ER resident proteins and proteins that are destined to other compartments, enter the ER
Lecture 6 – The secretory pathway: vesicular transport
Quality control in the ER (I)
Proteins synthesized in ER are checked by a chaperone for accurate folding without abnormalities so
that only proteins with normal folding are transported to the Golgi apparatus.
Lecture 6 – The secretory pathway: vesicular transport
Quality control in the ER (II)
Calnexin and Calreticulin work with two enzymes to repair unfolded proteins. These enzymes are:
a)
Glucosidase
b)
Glycosyl-transferase
They achieve this by the following pathway:
1) Glucosidase I removes the terminal glucose from the unfolded protein
2) Glucosidase II removes the second glucose
3) This allows for binding of the membrane bound Calnexin (or Calreticulin) which induces protein folding
4) Glucosidase II removes the final glucose
5) If the protein is now folded correctly it can exit from the ER
6) If the protein is misfolded, it interacts with soluble Calreticulin and then glucosyl-transferase adds three
new Glucose residues onto the protein, meaning it is passed around the cycle once more.
Lecture 6 – The secretory pathway: vesicular transport
Proteins synthesized in ER are folded with the aid of a chaperone. Accurate and complete folding of the
completed protein is also confirmed. Proteins not completely folded are stopped and folding is once
again attempted. This is because abnormal proteins occur when folding is inaccurate or incomplete,
which can lead to abnormal cellular functions or aggregate formation. Proteins that have been folded are
then transported to the Golgi body as part of the next step. However, proteins that could not be
successfully folded because of some sort of abnormality are transported from ER to the cytoplasm,
where they are degraded by a protein degradation apparatus called the proteasome. This series of
reactions is called protein quality control.
If the protein is properly folded, it can continue its journey into the Golgi.
Lecture 6 – The secretory pathway: vesicular transport
The secretory pathway is a transport system that shuttles macromolecules between the different
compartments in a cell. Movement between intracellular compartments is carried out by transport
vesicles (coated vesicles) that bud from one compartment and fuse with another in a highly organized
manner allowing movement of cargo in a unidirectional, prescribed pattern.
Which are the functions of the coats (cages of proteins)?
1) Protein concentration
2) Shaping of the vesicle
Which vesicular coats have been identified so far?
1) Clathrin-coated vesicles
2) COP-I-coated vesicles
3) COP-II-coated vesicles
They are present in different compartments
Lecture 6 – The secretory pathway: vesicular transport
How do vesicles find their right destination?
Membrane fusion requires bringing two
lipid membranes to within 1.5 nm of
each other so that they can join
• Water must be displaced from the
hydrophilic surface of the
membrane – energetically highly
unfavorable
Rab proteins (GTPases) on the surface of the vesicles are recognized by specific proteins on the
surface of the target membrane
v-SNARE proteins on the surface of the vesicles mediate fusion of the 2 lipid bilayers (of the vesicle
and of the target membrane) through interaction with t-SNARE proteins on the target membrane
(SNARE proteins mediate membrane fusion)
Lecture 6 – The secretory pathway: vesicular transport
What does it happen to resident ER proteins that escape in the Golgi?
Retrograde transport to the ER
1) Soluble resident proteins of the ER contain the tetrapeptide KDEL at the C-terminus. The KDEL signal
of the escaped ER proteins is recognized and bound by receptors in the Golgi. Binding triggers the
return of the KDEL proteins by retrograde vesicular transport. In the ER, the retrieved proteins
dissociate and the unoccupied KDEL receptors return to the Golgi.
2) Many ER transmembrane proteins, on the other hand, contain a
dilysine (KKXX) motif at their C-terminus cytosolic tail. This is
also a retrieval signal that allows recognition and subsequent
retrograde transport.
Lecture 6 – The secretory pathway: vesicular transport
Trans Golgi Network (TGN)
Different protein destinations:
1) Secretory storage granules
2) Cell surface
3) Endosomes
4) Lysosomes
5) Back to Golgi
Lecture 6 – The secretory pathway: vesicular transport
Ways macromolecules enter the cell
Endocytosis: 3 types
Phagocytosis
Pinocytosis
Process by which certain cells called
phagocytes ingest or engulf other
cells or particles. In higher animals,
phagocytosis is mainly a defensive
reaction against infection and invasion
of the body by foreign substances
(antigens).
Internalization of
extracellular fluid
Receptor-mediated
endocytosis
Import of specific
extracellular
macromolecules
(e.g. cholesterol)
All lectures: possible questions
 Which are the main features of lipid bilayers?
 Describe the features of pumps, channel and carriers.
 How can cells be visualized with fluorescence? Which are the characteristics of good
dyes?
 How is an action potential generated and propagated?
 What is myelin?
 Explain the differences between a prokaryotic and eukaryotic cell
 Which are the three main transport mechanisms?
 How are proteins targeted to the right location?
 Describe the structure of the nuclear pore
 How is the directionality of the nuclear transport maintained?
 Post-translational and co-translational ER transport. Explain
 Mitochondria and chloroplasts: similarities and differences
 Which are the principal energy sources for the translocation into mitochondria?
 Explain the main functions of ER and Golgi
 Which is the main function of calreticulin and calnexin proteins?
 What does mediate the recognition of a transport vesicle by the target membrane?
 Explain the functions of vesicle coating and which are the three types of coating
 What is the retrograde transport?
 How can macromolecules enter the cell?