FINAL Flashcards
1
Q
what are two organelles that are specific to animal cells?
A
- extracellular matrix: specialised material outside the cell
- lysosome: degradation of cellular components that are no longer needed
1
Q
what are three organelles that are not found in animal cells but are found in plant cells and some other cells?
A
- cell wall
- vacuole (2 types)
- chloroplast
2
Q
cell wall two functions
A
- cell shape
- protection against mechanical stress
3
Q
vacuoles two functions
A
- degradation (like animal lysosome)
- storage (small molecules and proteins)
4
Q
chloroplast function
A
- site of photosynthesis
5
Q
distinguish between the cytoplasm, the cytosol, and the lumen
A
- cytoplasm: contents of the cell outside the nucleus (membrane-bound organelles)
- cytosol: aqueous part of the cytoplasm. does not include membrane-bound organelles, does include ribosomes and cytoskeleton
- lumen: inside of organelles
6
Q
what cellular functions occur at membranes?
A
- compartmentalisation
- scaffold for biochemical activities
- selectively permeable barrier
- transport solutes
- respond to external signals
- interactions between cells
7
Q
describe the model proposed by Singer and Nicolson in 1972
A
Fluid Mosaic Model of the Membrane
- fluid: due to mobility of lipids and some of the proteins
- mosaic: many different lipids and many different proteins
- lipid bilayer: = 1 membrane consisting of two layers of leaflets
8
Q
define amphipathic molecules
A
have different biochemical/biophysical properties on different sides of the molecule
9
Q
what makes phospholipid molecules amphipathic?
A
they have a hydrophilic/polar head and hydrophobic tails
10
Q
state three types of lipids that membranes are composed of
A
- phospholipids
- sterols
- glycolipids
all have hydrophilic heads as well as hydrophobic tails
11
Q
phospholipids
A
- there are different types of membrane phospholipids
- most have a glycerol group (termed phosphoglycerides, of which there are different types)
12
Q
general structure of a phospholipid
A
polar head group (hydrophilic):
- different groups
- phosphate
glycerol
hydrocarbon tails
- length: 14-24 carbon atoms
- saturated/unsaturated
13
Q
kink
A
- hydrocarbon tail is unsaturated
- contains a cis-double bond
- this causes a bend in the tail
14
Q
what happens to phospholipids in aqueous environments?
A
- they spontaneously self-associate into a bilayer
- the polar head group interacts with water
- the two hydrophobic hydrocarbon tails interact with other hydrophobic tails
15
Q
how are sealed compartments formed by phospholipid bilayers?
A
- a planar phospholipid bilayer is energetically unfavourable as the hydrophobic tails are exposed to water along the edges)
- the formation of a sealed compartment shields hydrophobic tails from water
16
Q
define liposomes and describe their uses
A
artificial lipid bilayers used to:
1. study lipid properties
2. study membrane protein properties
3. drug delivery into cells (nanotechnology)
17
Q
how can membrane fluidity be visualised?
A
live cell imaging where laser tweezers are used to manipulate the membrane show that a membrane can be deformed without causing damage
18
Q
describe the different types of phospholipid movement within cell membranes
A
phospholipids within each leaflet rapidly:
- diffuse laterally (side-to-side or deeper into the membrane plane)
- rotate
- flex
- RARELY move from one leaflet to other (flip-flop) on their own
19
Q
why is cell membrane fluidity carefully regulated?
A
as it is important for function, e.g. membrane proteins for transport, enzyme activity, signaling
20
Q
two factors affecting membrane fluidity
A
- temperature
- composition (phospholipid saturation, phospholipid tail length, lipid composition)
21
Q
how does temperature impact membrane fluidity?
A
lower temperatures make the membrane more viscous and less fluid, which is unwanted
22
Q
how does composition - phospholipid saturation - impact membrane fluidity?
A
- cis double bonds increase fluidity at lower temperatures (reduce tight packing)
- phospholipids can change from being saturated to unsaturated to alter fluidity
23
Q
how does composition - phospholipid tail length - impact membrane fluidity?
A
shorter hydrocarbon tails increase fluidity at lower temperatures (lipid tails interact less)
24
how does composition - lipid composition - impact membrane fluidity?
the addition of cholesterol in animal cell membranes stiffens the membrane, making it less permeable to water
25
how are sterols typically present in animals and plants?
in animals, mainly cholesterol; in plants, plant sterols and some cholesterol
26
general structure of a sterol
- polar head group
- non polar rigid planar steroid ring structure
- non polar hydrocarbon tail
27
how does the addition of cholesterol molecules impact cell membranes?
- decreases the mobility of phospholipid tails (stiffens and thickens the membrane by filling space)
- plasma membrane is less permeable to polar molecules
28
draw a diagram of a cholesterol molecule surrounded by two phospholipid molecules
polar head
cholesterol-stiffened region
more fluid region
29
describe how lipid movement to the other leaflet is created in the ER membrane
- phospholipid synthesis adds to the cytosolic half of the bilayer
- scramblase (a phospholipid translocator in the ER membrane) catalyses the rapid flip lop of random phospholipids from one leaflet to another
- this ensures symmetric growth of both halves of the bilayer
30
distribution of phospholipids in the ER membrane is
random
31
how does distribution of phospholipids and glycolipids in the cell membrane differ from that in the ER?
the noncytosolic face and the cytosolic face have different lipids
- glycolipids only on noncytosolic face
- phosphatidylserine only on cytosolic face
32
how do phospholipids and glycoproteins end up on the plasma membrane?
- synthesised in the membrane of the ER
- carried in vesicles to the membrane of the Golgi apparatus
- carried in vesicles to the plasma membrane
33
how is the orientation of the cell membrane different to that of the membranes in the cytosol?
- the plasma membrane has the noncytosolic face towards the extracellular fluid and the cytosolic face towards the intracellular fluid
- the membranes in the cytosol (vesicles, Golgi, ER) have the cytosolic face toward the cytosol and the noncytosolic facing inside the organelle
34
describe the role of the Golgi membrane in regulation lipid membrane distribution
- delivery of new membrane from ER
- Flippase enzymes in the Golgi membrane catalyse the rapid flip-flop of specific phospholipids to the cytosolic leaflet (eg phosphatidylserine)
- some can bind cytosolic proteins at the plasma membrane (eg phosphatidylserine binds protein kinase c)
35
how are glycolipids and glycoproteins distributed on the membrane?
- formed by adding sugar groups to lipids/proteins on the luminal face of Golgi
- end up on plasma membrane, inside of organelles, on the noncytosolic face only
- protect the membrane from harsh environments
36
can proteins flip-flop?
no
37
two main subsets of membrane proteins
integral membrane proteins
peripheral membrane proteins
38
three types of integral membrane proteins
- transmembrane (cross the entire membrane, once or multiple times)
- monolayer associated (insert halfway)
- lipid-linked (have a lipid anchor inside the membrane)
39
peripheral membrane proteins
proteins do not insert into the membrane on either face of the membrane
- bound to other proteins or lipids by non-covalent interactions
40
distinguish between the extraction methods that need to be used to isolate integral membrane proteins as opposed to peripheral membrane proteins
integral: extraction methods use detergents (lipid bilayer destroyed)
peripheral: gentle extraction methods used (lipid bilayer remains intact)
41
describe the amphipathic properties of transmembrane proteins
amphipathic
- hydrophilic domains in aqueous environment (AA side chains polar)
- hydrophobic membrane-spanning domains (AA side chains non-polar)
42
what are the three main types of transmembrane proteins?
1. single alpha helix (single-pass)
2. multiple alpha helices (multipass)
3. beta barrel (rolled beta sheet, multipass)
43
how many amino acids long would you expect a membrane-spanning alpha helix to be?
20-30
44
draw a single alpha helix
slide 32
45
draw multiple alpha helices
slide 32
46
draw a beta barrel
slide 32
47
why is it important for transmembrane proteins to not be able to flip flop?
each transmembrane protein has a specific orientation - essential for function
48
how are the structures of transmembrane proteins identified?
1. X-ray crystallography: determines the 3D structure
2. hydrophobicity plots: hydropathy index, where +ve ΔG means more hydrophobic, -ve ΔG means mood hydrophilic index
49
describe mono-layer associated membrane proteins
proteins anchored on the cytosolic face by an amphipathic alpha helix
eg proteins in membrane bending for vesicle budding (Sar1) at the ER
50
describe two types of lipid-linked membrane proteins
1. protein with a GPI anchor (glycosylphosphatidylinositol). Synthesised in the ER lumen and ends up on the cell surface (noncytosolic face throughout whole process)
2. protein with another lipid anchor (fatty acid, prenyl). Cytosolic enzymes add the anchor, which directs the protein to the cytosolic face
51
Techniques: extraction of membrane proteins with detergents
Triton X-100 (has both hydrophobic and hydrophilic region)
By mixing the transmembrane protein with amphipathic detergent monomers and water, you form water-soluble protein-lipid-detergent complexes and water-soluble lipid-detergent micelles, which breaks up the membrane.
52
Technique: studying the properties of membrane proteins
Following the purification of the protein of interest, you can add phospholipids and remove the detergent to produce a functional protein incorporated into an artificial phospholipid vesicle. This can be used to study the properties of the protein in an isolated environment.
53
Technique: lateral diffusion of membrane proteins
- there is lateral diffusion of proteins within the leaflet, but no flip-flop
- study of protein movement can be done by Fluorescence Recovery After Photobleaching (FRAP), where the protein is fused to GFP (Green Fluorescent Protein)
54
describe how FRAP works
1. protein fused to GFP or labelled with fluorescent antibody
2. photobleach an area (white)
3. rate of fluorescence recovery: time taken for neighbouring unbleached fluorescent proteins to move into bleached area
55
look at the different possible graphs for FRAP on slide 43
56
can FRAP only be done with transmembrane proteins?
no, you can also do it with other proteins (eg cytosolic) and other molecules (eg lipids)
57
Permeability of an artificial bilayer as opposed to a cell membrane
Artificial bilayer (protein-free liposome): impermeable to most water-soluble molecules
Cell membrane: transports proteins to transfer specific molecules via facilitated transport
58
Movement across the lipid bilayer - small non polar molecules and small, uncharged polar molecules
Permeable - movement via simple diffusion through the lipid bilayer
1. high concentration to low concentration down the concentration gradient
2. more hydrophobic/nonpolar molecules have faster diffusion across the lipid bilayer
59
Small non-polar molecules
oxygen, carbon dioxide, nitrogen, steroid hormones
60
Small, uncharged polar molecules
water, ethanol - can diffuse across lipid bilayer
glycerol - cannot move across very well
61
Movement across the lipid bilayer - larger uncharged polar molecules and ions
Impermeable - require membrane proteins for transport
62
larger uncharged polar molecules
amino acids, nucleosides
glucose - a little can get across
63
Ions
H+, Na+, K+, Ca2+, Cl-, Mg2+, HCO3-
64
transmembrane transport proteins
- create a protein-lined hydrophilic path across cell membrane
- transport polar and charged molecules (amino acids, ions, sugars, nucleotides, various cell metabolites)
- each transport protein is selective and transports a specific class of molecules
- different cell membranes have a different complement of transport proteins
65
two main types of membrane transport proteins
- channels
- transporters
66
channel proteins vs transporter proteins in terms of selectivity and transport
channel proteins:
- selectivity: size and charge of electric solute
- transport: transient interactions as solute passes through. no conformational changes for transport through an open channel
transporter proteins:
- selectivity: solute fits into binding site
- transport: specific binding of solute. series of conformational changes for transport
67
passive transport
driven by the concentration gradient, no energy required
68
active transport
against concentration gradient, requiring energy
69
is glucose charged?
no
70
electrochemical gradient (electrical gradient) =
concentration gradient + membrane potential
71
what is the effect of the electrochemical gradient when the voltage and the concentration gradients work in the same direction?
- positive ions attracted to negative ions on the opposite side of the membrane (due to resting potential)
- this is additive to the effect of the concentration gradient
- greater net driving force
72
what is the effect of the electrochemical gradient when the voltage and the concentration gradients work in opposite directions?
- positive ions attracted to negative ions on the same side of the membrane (due to resting potential)
- smaller net driving force
73
describe voltage across a membrane
differences in charges of ions
74
resting membrane potential
stable electrical charge difference across a cell membrane when the cell is at rest and not actively sending signals
75
describe how channel proteins work
- hydrophilic pore across membrane
- most channel proteins are selective (eg ion channels transport a specific ion, determined by ion size and electric charge
- passive transport of solute
- transient interactions with channel wall as solute passes through (selectivity)
76
which are faster - channels or transporters?
channels
77
two broad categories of ion channels
- non-gated ion channels
- gated ion channels
78
non-gated ion channels
always open
79
gated ion channels
- some type of signal required for channel opening
- even when open, they are not open ALL the time, they just are open for more of the time
- specific ions are transported.
80
K+ leak channels
- non gated channel
- K+ moves out of cell
- major role in generating resting membrane potential in plasma membrane of animal cells
81
in what organisms are ion channels found?
animals, plants, microorganisms
82
types of gated ion channels
1. mechanically-gated
2. ligand-gated (extracellular ligand)
3. ligand-gated (intracellular ligand)
4. voltage-gated
83
mechanically-gated ion channels
signal - mechanical stress
eg plasma membrane may get stretched and that causes it to open up
84
ligand-gated (extracellular ligand)
signal - ligand from outside of the cell
eg neurotransmitter
85
ligand-gated (intracellular ligand)
signal - ligand from inside the cell
eg ion, nucleotide
86
what is the difference between the signal to open and the transported material?
the signal causes the channel to open, it is not necessarily what is transported through the channel
87
voltage-gated
signal - change in voltage across membrane by membrane depolarisation
88
main types of transporter proteins
1. passive transport by transporter proteins
- uniport
2. active transport by transporter proteins
- gradient-driven pumps (symport, antiport)
- ATP-driven pumps (P-type pump, V-type proton pump, ABC transporter)
89
how do transporter proteins work?
bind a specific solute
- goes through a conformational change to transport solute across the membrane
90
compare transporter proteins to channel proteins in terms of rate of transport by drawing graph
different kinetics - rate of transport in channels gets faster and faster as concentration difference of transported molecule increases. for transporter, because it has to undergo conformational changes, it starts off at a fast rate but eventually hits Vmax as all binding sites get saturated
91
uniport
one solute
- passive transport down its electrochemical gradient
- direction of transport is reversible - dependent on concentration gradient
92
glucose transporter
GLUT uniporter
- transports D-glucose down the concentration gradient
- can work in either direction (glucose in or out of the cell)
93
gradient-driven pump
- 1st solute down its gradient, providing energy
- 2nd solute against its gradient using this energy
94
ATP-driven pump (ATPases)
ATP hydrolysis provides energy to move the solute against its gradient
95
Light-driven pump (bacteria)
uses light energy to move solute against its gradient
96
two types of gradient-driven pumps
symport and antiport
97
symport
two solutes moved in the same direction
98
antiport
two solutes moved in opposite direction
99
Na+ glucose symporter
- sodium going down its electrochemical gradient, providing energy
- glucose is going against its concentration gradient
- random oscillations between conformations which are reversible
100
occluded state
transporter closed - either occupied or empty
101
when do conformational changes in the Na+ glucose symporter occur?
both sites occupied: cooperative binding of Na+ and glucose
both sites empty: both Na+ and glucose dissociate
102
describe the process behind the Na+ glucose symporter
* The symporter has two binding sites: one for Na+ and one for glucose.
* In its outward-open state, facing the extracellular space where Na+ concentration is high, Na+ readily binds to its site on the symporter.
* Once Na+ is bound, it increases the affinity of the transporter for glucose. When a glucose molecule binds to its site, this triggers a conformational change in the protein.
* The transporter then transitions to an occluded state where both binding sites are inaccessible from either side of the membrane. This occluded state can only be reached when both Na+ and glucose are bound or when neither is bound (occluded-empty).
* From this occluded state, another conformational change occurs that opens up towards the cytosol (inward-open state), where Na+ concentration is low.
* Due to this low intracellular concentration, Na+ dissociates from its binding site and enters into cytosol.
* The release of Na+ reduces affinity for glucose at its binding site; thus, glucose also dissociates and enters into cytosol.
103
how does Na+-H+ exchanger work
antiport: Na+ down its electrochemical gradient provides energy to move H+ against its electrochemical gradient
- sodium goes into the cytosol
- H+ goes out of the cell
104
what is the function of the Na+-H+ exchanger?
- cytosolic pH needs to be regulated for optimal enzyme function (pH~7.2)
- but excess H+ occurs in the cytosol from acid forming reactions, and leaks out of the lysosome
- transporters maintain cytosolic pH: when there is a drop in cytosolic pH, the transporter activity increases and H+ is transported out of the cell
105
how is the Na+ electrochemical gradient maintained in animal cells?
Na+-K+ pump (plasma membrane ATP-driven pump)
106
why does the Na+ electrochemical gradient need to be maintained?
- Na+ going down its electrochemical gradient provides energy for symport and antiport transporters
- continued action of gradient-driven pumps may equalise the Na+ gradient
107
P-type pumps
- use ATP
- phosphorylated during the pumping cycle
- many P-type pumps transport ions (H+, K+, Na+, Ca2+)
- flippases to transport phospholipids
108
Na+ and K+ moved ----- their electrochemical gradients
Na+ and K+ moved against their electrochemical gradients
109
sodium gradient used to:
- transport nutrients into cells (eg glucose)
- maintain pH
110
pumping cycle of the Na+-K+ pump
1. 3 Na+ bind from inside the cell
2. pump phosphorylates itself, hydrolysing ATP
3. phosphorylation triggers conformational change and Na+ is ejected
4. 2 K+ bind from the extracellular material
5. pump dephosphorylates itself
6. pump returns to original conformation and K+ is ejected
111
what do plant cells use instead of an Na+-K+ pump?
H+ pump
- generate H+ electrochemical gradient used for H+ driven symport/antiport
- leads to membrane potential
- H+ is moved from low (inside cell) to high (outside cell)
- solutes can then be moved into cell with H+
112
ABC transporters
use 2 ATP molecules to pump small molecules across the cell membrane
eg transport toxins outside of the cell but can also be source of chemotherapy resistance as cancer cells overproduce these transporters
113
V-type proton pump
- found in lysosome and plant vacuole
- uses ATP to pump H+ into organelles to acidify the lumen
114
F-type ATP synthase
- structurally related to V-type proton pump, but opposite mode of action
- uses H+ gradient (movement down) to drive the synthesis of ATP
- in mitochondria, chloroplasts, bacteria
115
compare a V-type proton pump to a F-type ATP synthase
V-type: uses ATP to pump H+ against the electrochemical gradient
F-type: uses the H+ electrochemical gradient to produce ATP.
116
is the action of the F-type ATP synthase reversible?
yes, it depends on ATP concentration and the H+ electrochemical gradient
117
how do transporters work together to transfer glucose from the intestine to the bloodstream?
epithelial cells of the villus
top of epithelial cells has microvilli - very high surface area
top of epithelial cell: apical domain
side: lateral domain
bottom: basal domain to face basal lamina
basal + lateral = bas-lateral domain
at top, we have Na+-glucose symporter which uses active transport to transport glucose against its gradient into the epithelial cell
in baso-lateral region, there is an Na+-K+ pump which keeps a low concentration of Na+ in the epithelial cell to maintain electrochemical gradient
GLUT uniporter carries out passive transport down the concentration gradient into extracellular fluid, which then ends up in the bloodstream
118
tight junctions between epithelial cells
creates a boundary where nothing can get by. proteins can move anywhere on apical surface or basal membrane but not past these junctions
helps keep transporters in their right compartment (eg GLUT uniporter and Na+-K+ pump stay on bottom, Na+-glucose symporter stays on top)
119
generation of membrane potentials
K+ leak channel (passive): facilitates outward flow of K+
1. K+ leak channels closed; plasma membrane potential = 0 (positive and negative charges balanced exactly)
2. K+ leak channels open; membrane potential exactly balances the tendency of K+ to leave
Na+-K+ pump
~10% of membrane potential
maintains:
- Na+ gradient with low cytosolic [Na+]
- K+ gradient with high cytosolic [K+]
electrogenic:
- 3Na+ pumped out
- 2K+ pumped in
- net 1+ ion pumped out
120
what is the usual balance in an animal cell?
cells generally balance electrical charges inside and outside of cell:
- extracellular space: high [Na+], high [Cl-], low [K+]
- cytosol: low [Na+], low[Cl-], high [K+], cell's fixed anions (nucleic acids, proteins, cell metabolites)
but:
- K+ flows out (K+ leak channels), ions diffusing from high to low
- Na+-K+ pump resulting in net 1+ ion pumped out
net result:
- bit more positive on outside (Na+, K+)
- bit more negative on inside (Cl- and fixed anions)
- forms membrane potentials
121
equilibrium =
resting membrane potential, which in animal cells varies from -20mV to -200mV
122
draw a diagram to show the generation of membrane potential in animal cells
123
generation of membrane potential in plant cells
plasma membrane P-type pump
- H+ pump
- generates H+ electrochemical gradient of -120 to -160mV
- used by gradient-driven pumps to carry out active transport
- electrical signaling
- regulating pH
124
the volumes taken up by organelles will...
differ for different cell types
125
percentage volume taken up by cytosol
- half the cell volume
- part site of protein synthesis and degradation
- where many metabolic pathways and cytoskeleton occur
126
rough ER
- over 50% of total cell membrane
- membrane-bound ribosomes
- synthesis of soluble proteins and transmembrane proteins for the endomembrane
127
smooth ER
- doesn't have membrane bound ribosomes
- phospholipid synthesis, detoxification
128
percentage of total cell membrane of rough ER membrane in liver hepatocyte vs pancreatic exocrine cell
60% in pancreas, 35% in liver
- pancreas has to synthesise enzymes.
129
why are mitochondria so abundant in liver hepatocytes?
provide energy to support the many metabolic functions on the liver
130
definition of an organelle
discrete structure or subcompartment of a eukaryotic cell that is specialised to carry out a particular function (most are membrane-enclosed)
131
how were organelles discovered?
via visualisation in a light or electron microscope
132
examples of membrane-enclosed organelles
- nucleus
- endoplasmic reticulum
- Golgi apparatus
133
examples of organelles that are not membrane-bound
- nucleolus
- centrosome
also known as bimolecular condensates
134
what is protein sorting?
- proteins are nuclear encoded
- mRNA arrives in cytoplasm and translation starts on ribosomes in cytosol
- cytosolic protein doesn't have a sorting signal, so its default location is the cytosol
- some proteins have a sorting signal called a signal sequence
135
what does a signal sequence consist of?
a stretch of amino acid sequence in a protein which directs the protein to the correct compartment
136
each signal sequence specifies
a specific destination in the cell; specific signal sequences direct proteins to nucleus, mitochondria, ER, peroxisomes, etc
137
signal sequences are recognised by
sorting receptors that take proteins to their destination
138
post-translational protein sorting
- proteins are nuclear-encoded
- fully synthesised in cytosol before sorting
- folded: nucleus, peroxisomes
- unfolded: mitochondria, plastids
139
co-translational sorting
- proteins are nuclear-encoded
- they have an ER signal sequence and are associated with ER during protein synthesis in the cytosol
140
proteins that are intended for the nucleus have a
nuclear localisation signal (NLS) for import into the nucleus.
141
function of the nuclear import receptors
nuclear import receptor (sorting receptor) binds the NLS and move it into the nucleus. nuclear pores act as gates to the nucleus - proteins with the nuclear import receptor are recognised.
142
function of transcription activators
required in the nucleus for eukaryotic transcription - imported through the nuclear pore and binds to DNA to bind to the activated target gene
143
function of peroxisomes
contain enzymes for oxidative reactions
- detoxify toxins, break down fatty acid molecules
144
enzymes imported into the peroxisome through
a transmembrane complex - there is a peroxisomal import receptor (sorting receptor) which binds to the peroxisome import sequence (signal sequence)
145
how and why do proteins have to move into mitochondria/chloroplasts
- have own genomes and ribosomes
- but most proteins for these organelles are nuclear-encoded
- translated in cytosol and targeted by a signal sequence for import
- proteins are unfolded for import by association with hsp70 chaperone proteins
146
transmembrane complexes in mitochondria
needs proteins to be unfolded to pass through. signal sequence bound to sorting receptor, which brings it to transmembrane complexes - hsp70 proteins come off as the protein moves through. then the mitochondrial hsp70 binds to it in the mitochondrial matrix (these help the protein fold and remove the signal)
147
why do proteins sort to the ER?
entry point to endomembrane system (ER, Golgi, endossâmes, lysosomes, or up to plasma membrane)
148
is ER signal hydrophobic or hydrophilic?
hydrophobic
149
what is co-translational translocation?
insertion of protein into ER starts as translation continues (whole ribosomal complex moves together)
150
ER signal sequence is usually at
N-terminus
151
types of proteins entering the ER:
1. soluble proteins
2. transmembrane proteins (eg channel)
152
are ribosomes specific to cytosol/ER?
no; once they are done translating protein, they move back to the cytosol and pick up any protein (no specificity)
153
describe the process for co-translational translocation of a soluble protein
1. translation starts, N-terminal ER signal sequence emerges
2. recognised by SRP, elongation arrest by SRP
3. SRP-ribosome complex binds to SRP receptor and moves it to the translocon
4. translocon opens
5. protein synthesis resumes with protein transfer into ER lumen
6. signal peptidase cleaves ER signal sequence, which is hydrophobic - in lipid bilayer
7. protein released into ER lumen
8. translocon closes
154
destination of soluble protein
lumen of an endomembrane organelle or secretion at PM
155
co-translational translocation for transmembrane protein (N-terminal ER signal sequence)
1. translation starts, N-terminal ER signal sequence emerges
2. recognised by SRP, elongation arrest by SRP
3. SRP-ribosome complex binds to SRP receptor which moves it to the translocon
4. translocon opens
5. protein synthesis resumes with protein transfer into ER lumen
6. stop-transfer sequence, which has an internal hydrophobic segment (single-pass transmembrane protein w a membrane spanning alpha helix) enters translocon
7. protein transfer stops and transmembrane domain released into lipid bilayer
8. signal peptidase cleaves ER signal sequence and translocon closes
9. N-terminus is in the ER lumen, C-terminus is in the cytosol, hydrophobic sequence is in it
156
translocon
protein channel complex in the endoplasmic reticulum (ER) membrane that facilitates the translocation of proteins across or into the ER membrane
157
destination of transmembrane protein
membrane of an endomembrane organelle or in the plasma membrane
158
N-terminal ER signal sequence
- stretch of hydrophobic amino acids at N-terminus of protein
- removed by signal peptidase
159
Internal ER signal sequence
- stretch of hydrophobic amino acids (start-transfer sequence)
- not removed - remains part of protein = membrane spanning alpha helix
- In double-pass or multi-pass transmembrane proteins, these internal sequences work with stop-transfer sequences to embed multiple segments of polypeptides within membranes.
160
similarity and differences between N-terminal and internal ER signal sequence
both types of sequences direct proteins to enter or integrate into the ER membrane via similar mechanisms involving SRP recognition and targeting to translocons, their positions within polypeptides and subsequent fates differ.
161
differences between hydrophobic stop transfer and start transfer sequences
no differences other than order in the protein
162
co-translational translocation for transmembrane protein (internal ER signal sequence)
1. translation starts, internal start-transfer sequence emerges
2. recognised by SRP, elongation arrest by SRP
3. SRP-ribosome complex binds to SRP receptor which moves it to the translocon
4. translocon opens
5. protein synthesis resumes with protein transfer into ER lumen
6. stop-transfer sequence enters translocon
7. protein transfer stops
8. start-transfer sequence and stop-transfer sequence (internal hydrophobic segments = membrane spanning alpha helices) are released into lipid bilayer
9. translocon closes
163
what components do intracellular compartments in the endomembrane system exchange?
lipids and proteins
164
secretory pathway
proteins and lipids made in the ER are delivered to other compartments
- ER to outside (exocytosis)
- ER to lysosomes (via endosomes)
165
endocytic pathway
contents moved into the cell (endocytosis)
166
retrieval pathway
retrieval of lipids/selected proteins for reuse
167
what happens to the individual leaflets of the membranes during endo/exocytosis?
maintain their orientation throughout
168
vesicular transport
- vesicle: small, membrane-enclosed organelle in cytoplasm of a eukaryotic cell
- shuttle components back an forth in the endomembrane system (eg ER to Golgi)
- two main components: soluble proteins inside the vesicle and transmembrane proteins embedded in membrane of vesicle
169
cargo protein receptors
receptors can be used to select for cargo proteins and then released into the ER lumen
170
constitutive exocytosis pathway
- in all eukaryotic cells
- continual delivery of proteins (transmembrane, soluble) and lipids to plasma membrane
- includes constitutive secretion of soluble proteins (eg collagen for ECM)
171
regulated exocytosis pathway
- regulated secretion - in specialised cells
- stored in specialised secretory vesicles
- extracellular signal leas to vesicle fusion with plasma membrane and contents are released
- eg pancreatic β cells (insulin release with increased blood glucose)
172
path of a secreted protein from translation to plasma membrane
Translation starts on cytosolic ribosomes
* ER Signal sequence at N-terminus directs protein to ER
Co-translational translocation at ER
* Protein inserted through ER membrane by a translocon protein
* ER Signal sequence cleaved and left behind in ER membrane
* Secreted protein ends up in ER lumen
Secreted protein
* Moves in transport vesicles through secretory pathway (ER → Golgi apparatus → Plasma membrane)
* Vesicle membrane fuses with plasma membrane during exocytosis
* Secreted protein released to extracellular spac
173
Path of a transmembrane protein from translation to plasma membrane
Translation starts on cytosolic ribosomes
* ER Signal sequence (N-terminal or Internal) directs protein to ER
Co-translational translocation at ER
* Protein inserted through ER membrane by a translocon protein
* There are different ways that a transmembrane protein can be
inserted into ER membrane
Transmembrane protein
* Moves in transport vesicles through secretory pathway (ER → Golgi apparatus → Plasma membrane)
* Vesicle membrane fuses with plasma membrane during exocytosis
* Transmembrane protein transferred to plasma membrane
174
how is maintenance of membrane protein asymmetry maintained?
- each membrane protein has a specific orientation
- this is a result of membrane orientation in the ER
- this protein symmetry is maintained through vesicular transport
175
Golgi function
receives proteins and lipids from the ER, modifies them, and then dispatches them to other destinations in the cell
176
Golgi structure
stack of flattened membrane-enclosed stacks (cisternae)
enters via:
- cis Golgi network
- cis cisternae
- medial cisterna
- trans cisterna
- trans Golgi network
leaves
177
animal vs plant cell Golgi
animal cells tend to have one bind Golgi structure; plant cells have a lot of smaller Golgi spread out throughout the cell
178
protein glycosylation
1. starts in the ER
2. a single type of oligosaccharide is attached to many proteins
3. Complex oligosaccharide processing occurs in the Golgi apparatus (a multistage processing unit - different enzymes in each cisterna)
4. glycosylation modifications for proteins and lipids (glycosylated lipids and proteins end up on the outside of cell to protect membrane + proteins from damage)
179
endocytic pathway: endosomes and lysosomes
digest material that is no longer needed by the cell
180
Endosomes
- membrane-bound organelles
- contain material ingested by endocytosis
1. endocytic vesicles fuse to early endosomes and ingested material sorted so that it will either go to the Golgi (recycling pathway) or to lysosomes (degradation pathway)
Degradation:
2. early endosomes mature into late endosomes
3. lysosomal proteins (hydrolases, H+ pump) continue to be delivered from trans Golgi network to either late or early endosomes
4. late endosomes mature into lysosomes
181
lysosomes
- membrane-bound organelles
- contain hydrolytic enzymes to digest worn-out proteins, organelles, other wast
182
difference between early and late endoscope
late not recycling cargo anymore, committed to cargo degradation pathway
183
where does digestion start?
at the late endosome
184
how does trans Golgi know that it needs to package up lysosomal proteins into vesicles to send them to vesicles?
specific types of sugar groups can be added to vesicles that are signals
185
types of enzymes contained in lysosomes
around 40 types of hydrolytic enzymes known as acid hydrolases (proteases, nucleases, lipases, etc)
186
lysosomes are acidified by
- H+ pump (V-type ATPase)
- low pH needed for hydrolytic enzymes
187
how does the lysosome protect the rest of the cell from digestion?
- it is membrane bound
- lysosomal membrane proteins (noncytosolic face, inside) are glycosylated for protection from proteases
188
function of transport proteins in lysosomal membrane
transfer digested products to cytosol (amino acids, sugars, nucleotides)
189
why do cytosolic proteins not have signal sequences?
as they stay in the cytosol`
190
3 different pathways of protein movement
secretory pathway
endocytic pathway
retrieval pathway
191
what gives vesicles directionality?
- directed movement of transport vesicles
- pulled by motor proteins associated with cytoskeleton
192
define the cytoskeleton
a highly dynamic network of proteins with many important functions
193
four main roles of the cytoskeleton
1. structural support (AF, MT, IF) for cell shape
2. internal organization of cell (MT) for organelles and vesicle transport
3. cell division (AF, MT) for chromosome segregation and division of cell into 2
4. large scale movements (AF) - crawling cell and muscle contraction
194
three components of cytoskeleton
actin filaments (d:~7nm), microtubules (d:~25nm), intermediate filaments (d:~10nm)
195
range of diameter of cytoskeletal filaments
7-25nm
196
light microscopy
- resolution limit of ~200nm
- limits from wavelength of visible light
- cannot resolve cytoskeletal filaments
197
fluorescence microscope
- light microscope with same resolution
- but fluorescent labels are added to detect specific proteins (eg cytoskeletal filaments)
198
transmission electron microscope
- uses beams of electrons of very short wavelength
- resolution limit of ~1nm
- reveals detailed structures
199
immunofluorescence microscopy
- used to determine location of proteins within cell
- cells are fixed (not light imagine)
- primary antibody used to bind to specific protein of interest
- secondary antibody binds to the primary antibody covalently tagged to a fluorescence marker
- fluorescence microscope used to excite fluorescent marker and visualise light emitted
200
draw a simplified diagram of the three types of filaments
201
filaments are held together by
noncovalent interactions
202
intermediate filaments
- involved in structural support
- different types of IF proteins
203
two main types of IFs
cytoplasmic and nuclear
204
cytoplasmic IFs
- in animal cells subjected to mechanical stress
- provide mechanical strength
205
nuclear IFs
- nuclear lamina - 2D meshwork formed by lamina in all animal cells
- plants have different lamin-like proteins
206
do plants need cytoplasmic IFs?
no; the cell wall provides most of the mechanical strength
207
describe the structure of cytoplasmic intermediate filaments
1. Proteins:
- conserved α-helical central rod domain
- N- and C- terminal domains differ
2. Pack together into rope-like filaments
- 2 monomers → coiled-coil dimer
- 2 dimers → staggered antiparallel tetramer
- 8 tetramers associate side by side and
assemble into filament
- most interactions are noncovalent
* No filament polarity - because no polarity in
tetramer (ends are the same)
* Tough, flexible, high tensile strength
208
Give an example of intermediate filaments
Keratin filaments in epithelial cells
- forms network throughout cytoplasm out to cell periphery
- anchored in each cell at cell-cell junction (desmosomes) and connect to neighbouring cells
- provide mechanical strength
209
define an epithelium
sheet of cells covering an external surface or lining an internal body cavity
210
function of microtubules
- cell organization: vesicle transport, organelle transport and positioning, centrosome in animal cells
- mitosis
- structural support for cells and motile structures (flagella, cilia)
211
structure of microtubules
- Long hollow tubes made of individual subunits of two closely related globular proteins, α-tubulin and β-tubulin
- form a tubulin heterodimer bound to GTP
- This regular arrangement of α & β subunits gives the microtubule polarity (plus end (β) is different from minus end (α))
- 13 parallel protofilaments make up a hollow tube
212
all bonds between individual subunits of microtubule profilaments are
noncovalent
213
the bonds between protofilaments are ---- than the bonds within each protofilament
weaker
214
can growth and disassembly of microtubules can occur at both ends?
yes, but is more rapid at plus end
215
experiment to show that microtubule growth is faster at the plus end
1. A bundle of microtubules isolated from a cilium
2. Isolated microtubules incubated with a high concentration of tubulin (subunit) and GTP
3. Faster growth of microtubules (more heterodimers being added) at the plus end
216
dynamic instability
plus ends of microtubules grow and shrink, which is needed for remodelling
217
dynamic instability: growing
1. free αβ-tubulin dimers bound to GTP are added to growing microtubule at plus end
(minus end stabilized at MTOC)
2. Shortly after dimer added to microtubule, β-tubulin hydrolyzes GTP to GDP
3. there is rapid addition of αβ-tubulin dimers which is faster than GTP hydrolysis in newly
added αβ-tubulin dimers
4. this leads to formation of GTP cap which stabilizes plus end
5. Microtubule continues to grow
218
dynamic instability: shrinking
1. free αβ-tubulin dimers bound to GTP are added to growing microtubule at plus end
(minus end stabilized at MTOC)
2. Shortly after dimer added to microtubule, β-tubulin hydrolyzes GTP to GDP
3. there is slower addition of αβ-tubulin dimers which is slower than GTP hydrolysis in newly added αβ-tubulin dimers
4. this leads to the GTP cap being lost, so now there is GDP-tubulin at plus end which has weaker binding
5. Microtubule disassembles
219
function of an MTOC
have nucleating sites for microtubule growth to start assembling new microtubules
eg centrosome in animal cells
220
example of a nucleation site
γ-Tubulin Ring Complex (γ-TuRC):
- protein complex of γ-tubulin & accessory proteins
- ring of γ-tubulin (gold) - acts as an attachment site for αβ-tubulin dimers
- minus end of microtubule at γ-TuRC
- plus end of microtubule grows out
221
does the alpha tubulin or beta bind to y tubulin
alpha
222
example of the dynamic nature of the MTOC (non dividing animal cells in interphase)
- mos microtubules radiate from one centrosome
223
example of the dynamic nature of the MTOC (dividing animal cells)
- centrosome duplicates to form two spindle poles (MTOCs)
- microtubules are reorganised to form a bipolar mitotic spindle, which requires microtubule dynamics (disassembly/assembly)
224
4 functions of microtubule-associated proteins
- nucleate growth of new microtubules
- promote microtubule polymerisation
- promote microtubule disassembly
- stabilize microtubules (prevent disassembly) by binding to the sides and plus-end linking the protein
225
Role of micro tubules in axons
- how do neurotransmitters synthesized in the ER get to the axon terminals?
- ER and Golgi apparatus are located in the nerve cell body
- these neurons can be a meter long: from your spinal cord to your fingertip
cargo transport from the cell body to the axon is done by motor proteins on microtubule
226
motor proteins for microtubules
kinesins and dyneins
227
kinesins
generally move towards plus end of microtubules
eg. kinesin I: towards plus end to axon terminus, cargo of organelles, vesicles, macromolecule
228
dyneins
generally move towards the minus end of microtubules
eg. cytoplasmic dynein: towards minus end to cell body, cargo of worn-out mitochondria and endocytosed materia
229
describe the dimeric structure of kinesin-1 and cytoplasmic dynein
- heads move along microtubules, use ATP hydrolysis for movement
- tails - transport cargo
230
where do microtubules position organelles?
microtubules go from the centrosome (MTOC) to cell periphery
the ER is pulled from the nuclear envelope to the cell periphery by kinesin-1 (towards microtubule plus end)
Golgi is held near the centrosome by cytoplasmic dynein (towards microtubule minus end)
231
actin filaments are also known as
microfilaments
232
arre actin filaments present in all eukaryote?
yes
233
what are actin filaments made of?
- actin monomers
- flexible, extensible
234
what motor proteins use actin filaments?
myosins
235
functions of actin filaments
1. stiff, stable structures (microvilli)
2. contractile activity
3. cell motility (crawling)
4. cytokinesis
236
structure of actin filaments
- helical filament composed of a single type of globular protein - actin monomers, which are held together by noncovalent interactions
- an actin filament is made by two protofilaments twisted in a right-handed helix
237
is an actin filament polar? explain
- plus end is different from minus end
- actin monomers all in the same orientation in each protofilament
- growth is faster at the plus end
238
what are free actin monomers bound to?
ATP, which is bound in the centre of the protein
239
how are actin monomers added to the filament?
- actin hydrolyses ATP to ADP
- reduces strength of binding between monomers in filament
- rapid addition of actin monomers
- this is faster than the ATP hydrolysis in newly added actin monomers, causing actin filament to have an ATP cap, stabilising the structure
240
actin polymerisation in a test tube (in vitro)
Actin subunits (monomers) and
ATP added to a test tube to study actin filament polymerization
Nucleation (lag phase):
* small oligomers form but are
unstable
Elongation (growth phase):
* some oligomers become more
stable, leads to rapid filament
elongation (faster at plus end)
Steady state (equilibrium phase):
* decrease in [actin subunits]
* rate of subunit addition = rate of
subunit disassociation
* length doesn't change
* Treadmilling
241
Process of actin filament growth
At the plus end, there is ATP-actin:
* addition of actin monomers - polymerization
* shortly after, actin hydrolyzes ATP → ADP
At the minus end, there is ADP-actin:
* loss of actin monomers - depolymerization
242
what happens at Treadmilling Concentration?
Actin filament remains the same size and looks “stable” but there is continual exchange of monomers at ends:
* net addition at the plus end
* net loss at the minus end
Actin monomers move through the filament
until they are eventually replaced
- continuous supply of ATP needed
243
cell crawling
- dynamic changes in actin filaments
- an example where actin filaments undergo treadmilling
- actin filaments must rapidly assemble at the leading edge (red) and disassemble further back to push the leading edge (and cell forward)
244
compare actin filaments to microtubules
245
what are the different functions of actin filaments regulated by?
actin binding proteins
246
6 examples of regulation by actin binding proteins
- sequester actin monomers (prevent polymerization)
- promote nucleation to form filaments
- stabilize actin filaments (capping)
- organize: bundle, cross-link filaments
- sever actin filaments
247
what do myosins generally do?
move towards plus end of actin filaments. their heads move along actin filaments, use ATP hydrolysis for movement
248
two types of myosin proteins
myosin I
myosin II
249
myosin I
tail domain: binds cargo
* e.g. (B) vesicles (regulated secretion)
* e.g. (C) plasma membrane (shape)
250
xmyosin II
dimer
* tails: organized in a coiled-coil
* dimers assemble into myosin-II filaments through their coiled-coil tails
* e.g. bipolar myosin-II filament, which slide actin filaments in opposite directions
(plus end of both actin filaments) and generates a contractile force
251
how do epithelial cells interact with each other and the extracellular matrix?
through junctions to form tissues
252
what are the 5 types of junctions?
tight junctions
adherens junction
desmosome
gap junction
hemidesmosome
253
draw a diagram labelling the positioning of the 5 different junctions
254
what types of junctions are present in epithelial cells?
all junctions
255
function of tight junction
- help polarise cells
- create a tight seal between cells, preventing mixing of the extracellular environment
- act as fences in the membrane, preventing mixing of apical and basolateral membrane proteins
256
adherens junction
joins an actin bundle in one cell to a similar bundle in a neighbouring cell, thus sticking 2 cells together
257
desmosome
joins the intermediate filaments in one cell to those in a neighbour cell, thus sticking 2 cells together
258
gap junction
allow for communication between cells:
- couple cells electrically and metabolically
- allow passage of ions and metabolites (<1000 daltons)
- not very selective as to what passes through
259
how does a hemidesmosome differ from an adherens junction and a desmosome?
it is a cell-ECM anchoring junction
260
why are mature epithelial cells polarised?
junctions are arranged in a specific order (ie ends are different
261
give an example of the polarity of epithelial cells
sealing strand (tight junction belt) above the adhesion belt
262
intercellular junctions in plant cells
- plant cells lack cell junctions found in animal cells
- they are surrounded by cell walls (hold cells together, provide mechanical strength)
- plasmodesmata are intercellular junctions that allow for communication between cells
- need to cross cell wall, so have different structure from gap junctions
263
tight junctions form
sealing strands (a tight junction belt)
264
tight junctions are composed of two transmembrane proteins:
Claudin and occludin
- required in both cells
- extracellular domain in one cell interacts with the extracellular domain in the neighbouring cells
- homophilic interactions (occludin attracted to occludin, claudin attracted to claudin)
265
adherens junctions, desmosomes, and hemidesmosomes are also termed
anchoring junctions
266
function of anchoring junctions
provide mechanical strength to the epithelium
267
function of cell-cell anchoring junctions
link cytoskeletons of neighbouring cells
268
function of cell-ECM anchoring junctions
link cytoskeleton to basal lamina
269
two types of proteins involved in anchoring junctions
adhesion proteins and linker proteins
270
transmembrane adhesion proteins
- transmembrane proteins
- extracellular domains interact with adhesion proteins of neighbouring cell (side) or extracellular matrix (bottom)
- intracellular domains interact with linker proteins
271
intracellular linker proteins
- cytosolic proteins
- link transmembrane adhesion proteins to cytoskeletal filaments
272
each anchoring junction has specific transmembrane adhesion proteins and intracellular linker proteins. give:
- transmembrane adhesion protein
- extracellular binding
- intracellular cytoskeletal attachment
adherens junction:
- classical cadherins
- classical cadherin on neighbouring cell
- actin filaments
desmosome:
- nonclassical cadherins (desmoglein, desmocollin)
- desmoglein and desmocollin on neighbouring cell
- intermediate filaments
hemidesmosome:
- α6β4 integral
- extracellular matrix proteins
- intermediate filaments
273
adherens junctions
- form an adhesion belt that encircles the inside of the plasma membrane
- transmembrane adhesion proteins = classical cadherins
- cadherin proteins from neighbouring cells interact with each other via homophilic interactions (eg e-cadherin/e-cadherin)
- intracellular linker proteins link cadherin proteins to actin filaments
- cadherin proteins become concentrated at sites of cell-cell interactions, forming adherens junctions
274
both desmosomes and hemidesmosomes link to ----; why?
intermediate filaments eg keratin filaments. intermediate filaments provide the most structural strength
275
distinguish between desmosomes and hemidesmosomes
- desmosomes are linked to keratin filaments and connect to a neighbouring cell
- hemidesmosomes anchor keratin filaments to the basal lamina
276
desmosomes
- transmembrane adhesion proteins = nonclassical cadherin proteins (desmoglein, desmocollin)
- these undergo homophilic and heterophilic binding
- intracellular linker proteins link desmoglein and desmocollin to keratin filaments inside the cell
277
hemidesmosomes
- transmembrane adhesion proteins = integrins that bind to laminin in the basal lamina (ECM)
- intracellular linker proteins link integrins to keratin filaments inside cell
278
describe the structure of a gap junction
1 subunit = connexin
6 connexins = form connexon (hemichannel), which by itself is closed
2 connexons = form intracellular channel (open)
279
passes through gap junctions:
cAMP, nucleotides, glucose, amino acids
280
does not pass through gap junctions:
macromolecules, proteins, nucleic acids
281
describe the gated nature of gap junctions
can be in an open or closed state by extracellular or intracellular signals
eg treatment with dopamine causes close gap junctions
282
dramatic increase in cytosolic Ca2+ ->
close gap junction
283
membrane damage ->
- Ca2+ leaks into the damaged cell
- gap junctions close
- prevents loss of metabolites from adjacent cells
284
describe the structure and functioning of the plasmodesmata
- cytoplasmic channels which lead to a continuous plasma membrane and ER across plasmodesmata
- intercellular free movement of soluble molecules (<1000 daltons), like sugars, ions, other essential nutrients
- controlled trafficking of larger soluble molecules via gating, like proteins or regulatory RNAs
285
callose deposition in cell wall
- callose is a plant polysaccharide
- permeability control through reversible callose deposition
286
animal tissues are composed of
cells and extracellular matrix
287
compare epithelial tissue and connective tissue
epithelial tissue (epithelium):
- eg intestinal lining, skin epidermis
- cells closely associated and attached to each other
- limited ECM (a thin basal lamina)
- cytoskeletal filaments provide resistance to mechanical stress
connective tissues:
- eg skin dermis, bone, tendon, cartilate
- cells are rarely connected and are attached to the matrix
- plentiful ECM
- ECM provides resistance to mechanical stress
288
what gives different tissues different properties?
different compositions of ECM
289
what is the primary component in connective tissues?
ECM
290
3 major classes of macromolecules in the extracellular matrix:
1. glycosaminoglycans (GAGs) and proteoglycans
2. fibrous proteins (collagens, elastin)
3. glycoproteins (eg laminin, fibronectin)
291
connective tissue ECM: glycosaminoglycans (GAGs)
- long, linear, chains of a repeating disaccharide
- highly negatively charged (attract Na+ and water)
- form hydrated gels, resist compression
- space filling
- most GAGs synthesised inside cell and released by exocytosis
292
hyaluronan
- simple GAG
- long chain of repeating disaccharide subunits (up to 25,000)
- hyaluronan is spun directly from cell surface by a plasma membrane enzyme complex
293
connective tissue ECM: proteoglycans
- subclass of glycoproteins
- protein with at least one sugar side chain which must be a glycosaminoglycan (GAG)
- typically, more extensive addition of sugars (up to 95% of total weight)
294
connective tissue ECM: collagen
- fibrous protein
- provides tensile strength
- resists stretching
295
structure of typical collagen (fibril-forming collagen)
- three chains wound around each other in a triple helix
- assemble into ordered polymers to form collagen fibrils, which can then pack together into collagen fibres
296
collagen is secreted as ----- by -----
procollagen by fibroblasts (skin, tendon, other connective tissue) and osteoblasts (bone)
297
once procollagen is secreted outside,
it is processed to collagen and assembled into large structures (collagen fibrils)
298
how do cells interact with collagen in the ECM of connective tissues?
- connective tissue cells that secrete collagen also organise collagen in the ECM
- they bind to collagen in ECM through integral (cell surface adhesion receptor) and fibronectin (glycoprotein)
299
fibronectin
- binds collagen
- binds integrin
300
integrin
- binds fibronectin (extracellular domain)
- binds adaptor proteins - actin filaments (intracellular domain)
301
connective tissue ECM: elastin
- elastin is a fibrous protein
- networks of elastin give tissues elasticity, allowing it to stretch and relax like a rubber band (resilience)
302
epithelial tissue ECM: basal lamina
the basal lamina is a basement membrane
- specialised type of ECM
- underlies all epithelia
- thin (40-120nm thick)
- ECM is secreted by the epithelial cells
- influences cell polarity (apical - basal)
303
how does the basal lamina separate the epithelia from underlying tissue?
- prevents fibroblasts in underlying connective tissue from interacting with epithelial cells
- yet allows passage of macrophages and lymphocytes
304
basal lamina contains a lot of
- laminin (glycoprotein)
- type 4 collagen (fibrous protein)
- integrin (transmembrane adhesion protein)
305
basal lamina is attachment site for
epithelia
306
basal lamina is anchored by
hemidesmosomes
307
basal lamina is organised by
laminin:
- interacts with other components of ECM
- links integrin to type IV collagen
308
describe the structure and contents of the plant cell wall
- more rigid than the ECM of animal tissues
- main components: cellulose, pectin (polysaccharides)
- cellulose microfibrils provide tensile strength
- pectin is space filling and provides resistance to compression
309
how is the plant cell wall made?
- plant cells synthesise cellulose chains at the plasma membrane using a cellulose synthase complex
- other cell wall components are synthesised in the Golgi and exported by exocytosis
310
cell cycle
- conserved in all eukaryotes
- sequence of events where contents of a cell are duplicated and divided into two
311
observing animal cell division in culture
- cells do not divide at the same time
- when cells do divide, all cells follow the same stages in mitosis
312
3 broad cell cycle stages conserved in all eukaryotes
1. cell growth and chromosome duplication
2. chromosome segregation
3. cell division
313
M phase
- nucleus and cytoplasm divide:
1. mitosis (nuclear division)
2. cytokinesis (cytoplasmic division)
314
interphase
period between cell divisions (metabolic activity, cell growth, repair)
- G1 phase
- S phase (synthesis)
- G2 phase
315
do all mature cells divide in multicellular organisms?
no; many mature cells do not divide
- eg terminally differentiated cells - nerve cells, muscle cells, red blood cells
- as they become differentiated, they lose the ability to divide
316
some cells only divide when given
an appropriate stimulus:
- eg when damaged, liver cells start to divide to replace damage tissue
317
examples of cells that normally divide on an ongoing basis
hematopoietic and epithelial stem cells
318
G0
cells that do not divide are in G0
- no cell division
- metabolically active, carry out cell function
319
3 checkpoints/transitions in the cell cycle
- start transition (G1 -> S)
- G2/M transition (G2 -> M)
- metaphase to anaphase transition (aka spindle assembly checkpoint)
320
what is the purpose of the cell-cycle control system?
to delay later events until the earlier events are complete
321
problems in checkpoints can cause
chromosome segregation defects
322
start transition
- decision to enter S phase
- is the environment favourable? eg sufficient nutrients, specific signal molecules
323
G2/M transition
- decision to enter mitosis
- is all DNA replicated? is all DNA damage repaired?
324
metaphase to anaphase transition?
- decision to pull duplicated chromosomes apart
- are all chromosomes properly attached to the mitotic spindle?
325
cell cycle progression is controlled by
molecular switches
326
how is entry into the next phase of the cell cycle ensured?
- triggered by cyclin-dependent protein kinases (Cdks)
- cyclin-Cdk complex is activated for entry, then inactivated (molecular switch)
327
entry into the M phase
M-Cdk (Idk activated by M cyclin) phosphorylates other regulatory proteins
328
how is entry into the next phase of the cell cycle paused?
by other regulators, if any answer to the questions is no:
- Cdk inhibitors block entry to the S phase
- inhibitio of activating phosphatase (Cdc25) blocks entry to mitosis
- inhibition of APC/C activation delays exit from mitos
329
interphase - G1 phase
centrosome duplication initiated and completed by G2
330
interphase - S phase
chromosomes replicated (decondensed)
- cohesions deposited to hold two sister chromatids together
331
interphase - G2 phase
- by the end of G2, the replicated chromosomes are dispersed and tangled
- need to reorganise and condense for mitosis
332
3 main steps of prophase
1. replicated chromosomes condense
2. centrosome duplication
3. mitotic spindle assembly
333
prophase - replicated chromosomes condense
chromosome condensation (chromatids compacted) and sister-chromatid resolution (separable units)
- cohesins removed from chromosome arms, but not from centromeres
- condensins condense DNA in each sister chromatid
- sister chromatids are resolved but remain associated at the centromere by cohesins
334
prophase - centrosome duplication
- centrosome duplicated once per cell cycle during interphase; initiated in G1 and completed by G2
- each centriole in the pair of centrioles in centrosome serves as a site for assembly of a new centriole
- duplicated centrosomes form poles of mitotic spindle
335
how are microtubules arranged in a non-dividing cell?
- in a radial pattern
- plus ends radiating out
- minus ends stabilised at the MTOC (centrosome)
336
what are the two conditions required for mitotic spindle assembly to start during prophase?
- disassembly and reassembly of microtubules (to go from radiating from a single centrosome to radiating from two)
- duplicated centrosomes
337
centrosome structure
- centrosome matrix surrounds the pair of centrioles
- contains y-tubulin ring complexes (y-TuRCs), which are nucleating sites to assemble new microtubules
338
pair of centrioles in the centrosome
- organised at right angles to each other
- composed of nine fibrils of three microtubules each
339
prophase - mitotic spindle assembly
- mitotic spindle assembly starts in prophase (M phase)
- requires microtubule dynamics (disassembly and assembly)
- duplicated centrosomes separate
- radial array of microtubules extend out from each to position centrosome, and will become the two spindle poles
340
nuclear envelope breakdown
occurs at the boundary between prophase and prometaphase
- phosphorylation of lamins and nuclear pore proteins triggers disassembly of nuclear envelope into small membrane vesicles
341
nuclear lamina
- meshwork of interconnected nuclear lamina proteins
- form a two dimensional lattice on the inner nuclear membrane
342
pro metaphase
- nuclear envelope is now disassembled
- mitotic spindle assembly can now be completed
- kinetochore microtubules in the mitotic spindle attach to duplicated chromosomes
- chromosome movement begins
343
mitotic spindle assembly and function requires
1. microtubule dynamics (disassembly and assembly)
2. microtubule motor protein activity (kinesics, cytoplasmic dynein)
344
state the whole order of the cell cycle
1. interphase (G1, S, G2 phase)
2. prophase
3. prometaphase
4. metaphase
5. anaphase
6. telophase
7. cytokinesis (NB: partly occurs alongside half of anaphase and telophase)
345
3 microtubules involved in the mitotic spindle
1. astra microtubules: anchored at the centrosome and help position the mitotic spindle. cytoplasmic dynein (motor protein) attached to PM and moves to minus ends, thus pulling centrosome towards PM
2. non-kinetochore microtubules: cross-linked microtubules throughout the mitotic spindle. microtubule-associated proteins (kinesin-5 and others) hold the microtubules together
3. kinetochore microtubules: attach duplicated chromosomes to the spindle poles
346
kinesin-5
walks towards the plus end of the non-kinetochore microtubules that it associates with, which have opposite orientations. this pushes the microtubules apart, pushing the centrosomes apart.
347
describe how kinetochore microtubules attach to chromosomes
- kinetochores are located at the centromeres of chromosomes
- there is one kinetochore for each sister chromatid in the duplicated chromosome
- microtubules from both spindle poles must attach to kinetochores of sister chromatids
- generates equal tension on both sides to line up chromosomes at equator of spindle
348
connecting protein complexes
- bind to sides of microtubule near plus end
- the exposed plus end of the microtubule allows for growing or shrinking for chromosome movement so it can be placed at the centre of the cell before separation
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metaphase
- all chromosomes are aligned on the metaphase plate (equator of the spindle)
- microtubule dynamos continue to maintain the metaphase spindle (tubulin flux)
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tubulin flux through microtubules
to maintain the metaphase spindle, there is a continuous:
- addition of tubulin subunits at plus end
- removal of tubulin subunits at minus end
- length of kinetochore microtubules does not change
this is an example of treadmilling
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how was tubulin flux confirmed?
- a small amount of fluorescent tubulin ('speckles') was added to observe microtubule flux
- a time lapse video microscopy was used to follow the fluorescent tubulin movement
- added at plus end
- depolymerises are removing the tubulin heterodimers from the minus end
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metaphase-anaphase transition (spindle assembly checkpoint)
anaphase does not start until all the chromosomes are aligned on the metaphase plate
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anaphase
separation of sister chromatids:
- separate activated, which then cleaves the cohesin complex
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anaphase A
- kinetochore microtubules are shortened (loss of tubulin at both ends) due to depolymerisation
- sister chromatids are pulled apart towards opposite poles
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anaphase B
- kinesin causes a sliding force between non-kinetochore microtubules from opposite poles, pushing the poles apart
- cytoplasmic dynein generates a pulling force at the cell cortex, dragging the two poles apart
- microtubule growth at the plus ends of non-kinetochore microtubules also helps push the poles apart
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telophase
- chromosomes are now separated into two groups, one at each spindle pole
- nuclear envelope reassembly takes place: there is dephospho rylation of nuclear pore proteins and lamins by phosphatase, causing the envelope lamina, and pores to reform
- mitotic spindle disassembles
- chromosomes decondense
- end of mitosis!
- contractile ring for cytokinesis is being assembled (starts in anaphase)
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cytokinesis in animal cells
- contractile ring divides the cytoplasm in two
- contractile force by contractile ring brings cell membrane in as contractile ring becomes smaller
- contractile ring disassembles once there are two daughter cells
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structure of contractile ring
assembled from actin and myosin filaments at the cleavage furrow (midway between spindle poles and underneath cell membrane)
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describe how actin and myosin II motor proteins are involved in causing contractile force
1. Actin filaments form a band at the division site.
2. Myosin II uses ATP to slide actin filaments towards each other; myosin moves toward plus end, pushing actin towards respective minus ends.
3. This sliding action constricts the ring and pulls in the membrane until cell division is complete
360
role of non-kinetochore microtubules in animal cell cytokinesis
send a signal to the plasma membrane at the plane of cleavage to say this is where we need the contractile ring to assemble
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how does mitosis in a plant cell differ from that in an animal cell?
- very similar but no centrosome (it has another mechanism to form the mitotic spindle)
- cytokinesis in the plant cell is different due to cell wall
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telophase in plant cells
- chromosomes separated into two sets
- phragmoplast starts to form
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what is the phragmoplast?
- specific structure to form cell plate
- has microtubules, actin filaments, vesicles from Golgi
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cytokinesis in plant cells
- nuclear envelope reassembled, chromosomes decondensed
- cell plate forms
- this is a transient membrane compartment (vesicles from Golgi fuse together) to divide into two
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G1 in plants
cell plate has matured into plasma membranes and cell wall between two daughter cells
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compare cell division in meiosis and mitosis
meiosis:
- one round of DNA replication (chromosome duplication)
- two rounds of cell division
- produces 4 haploid cells
- homologous chromosomes are paired at the metaphase plate
mitosis:
- one round of DNA replication (chromosome duplication)
- one round of cell division
- produces 2 diploid cells
- homologous chromosomes are not paired at the metaphase plate
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meiosis cell division rounds 1 and 2
Round 1: homologous chromosome are segregated into two daughter cells (sister chromatids remain attached)
Round 2: sister chromatids are segregated, producing 4 haploid cells
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give an example of how studying cell division in a multicellular organisms helps us understand the complexities of the cell cycle
biochemical studies:
- injecting cytoplasm from fertilised Xenopus eggs into Xenopus oocytes led to the discovery of cyclin-dependent protein kinases
