1
Q
what is immunofluorescence used for ?
A
used to visualise proteins and lipids in cells
movement of molecules can be visualised
2
Q
how does immunofluorescence work
A
- a fluorescent tag can be added to a primary or secondary antibody
- a primary antibody recognises a specific antigen
- secondary antibodies recognise primary antibodies from a particular specifies
3
Q
what does a scanning electron microscope do
A
provides the fine detail of the surfaces
4
Q
what is the resolution of a TEM
A
<0.1nm
5
Q
what is cryo-electron tomography
A
high resolution 3D imaging technique that uses an electron microscope to reconstruct the structure of frozen-hydrated biological samples
6
Q
what does cryo-electron tomography allow
A
it allows us to visualise protein complexes in their natural environment
7
Q
why is microscopy important
A
it allows us to validate our models based on biochemical or genetic interactions
it suggests new hypothesis
8
Q
how do prokaryotic cells arrange their interior
A
- no membrane bound organelle
- show segregation of their components within their cytoplasm
- the cytoskeleton helps to organise this segregation
9
Q
what are the cytoskeletal protein homologues in bacteria
A
Tubulin - FtsZ
Actin - MreB, MbI,ParM,MamK
intermediate filaments - CreS
10
Q
what does FtsZ do
A
controls cell division
11
Q
what does MreB ect. do
A
used for cell growth and shape
12
Q
what does CreS do
A
controls a curved shape
13
Q
similarities between fungi, plants and bacteria
A
- all have a cell wall
- Bacterial cell wall structure distinguishes Gram +ve and Gram -ve
14
Q
what additional protein coat do some bacteria have
A
S-Layer which is a two-dimensional crystalline array that
-is protective
-interacts with the host immune system
-acts as a molecular sieve
Also found in Archaea
15
Q
what are archaea
A
- single cell organism
- lack membrane bound nucleus
- many cellular functions differ from bacteria
- have an S-Layer
- tough cell wall so can grow in extreme envs
- not pathogens
16
Q
why is the cell wall important
A
- structural integrity
- maintenance of cell shape
- protection of osmotic lysis
- pathogenic
- major target for antibiotics
17
Q
plant cell walls
A
- largely composed of cellulose
- primary cell wall is tough but thin
- secondary cell wall is tougher with the addition of lignin
- maintain cell shape and integrity
- protection against osmotic lysis
18
Q
what is the function of the vacuole
A
- develops turgor pressure
- prevents wilting
- degradative functions
19
Q
fundamental properties of biological membranes
A
- barrier, selectively permiable
- flexible
- continuous
- self repairing
20
Q
major components of membranes
A
- lipids (phospholipids)
- proteins (contribute to function)
- sugars (on the outside, peptidoglycan)
21
Q
structure of phospholipids
A
- amphipathic
- hydrophobic and hydrophilic region
- can be cis or trans (only see cis in wild, ones that have the bend)
22
Q
what do phospholipids form in aq conds
A
- micelles or
- bilayers
23
Q
how do phospholipids move in the bilayer
A
they can rotate or exchange within the layer
or
move very slowly from one layer(leaflet) to the other
this rarely occurs spontaneously but does occur physiologically
24
Q
what does bilayer fluidity depend on
A
- composition
- number of double bonds in fatty acid chains
- length of fatty acid chains
- many and short = ^ fluidity
25
when are membranes fluid
- at the temp of the organism
- cold blooded animal membranes have more dbl bonds/shorter chains than warm blooded
26
what is phosphatidyl-serine
a negatively charged phospholipid
27
what does cholesterol do
- it makes membranes locally more rigid and less permeable
- it doesnt make the membrane more rigid at high concs of saturated fatty acid
- prevents the membrane from becoming crystalline
28
how do proteins associate with lipid bilayers
integral membrane proteins (embedded):
- transmembrane domains (e.g receptors)
- porins
- enzymes
peripheral membrane proteins:
- associate reversibly with membranes
29
biological membranes are not ...
homogenous
- they can form clusters or microdomains
30
what forms microdomains and why
cholesterol
so that proteins with longer transmembrane domains can cluster there
31
what is a type of membrane domain
apical and basolateral
- tight junctions prevent movement of proteins from one to another
32
why is membrane asymmetry important
- coagulation (blood clotting)
- recognition and clearance of dead cells
- blood groups (ABO system)
33
how do cells change shape
- membrane cytoskeleton interactions allow cells to change shape
- membranes are flexible
34
what effects the speed of membrane transport
- permeability co-efficient
- local temperature
- thickness
- conc gradients
- channels and carriers
35
how do membrane transport proteins and conc grads regulate transport rates
- each protein transports a particular solute
- solutes pass through the membrane without coming into contact with the hydrophobic core of the membrane
36
types of transport
passive - down a conc grad using channels and carriers, no energy needed
active - against a conc grad using carriers, uses energy
37
how else can transport be influenced
membrane potential
- voltage difference across membranes
combination of membrane potential and concentration gradient gives an electrochemical gradient
38
how do channel proteins work
- form weak interactions with solute
- rapid transport down conc gradient
- forms a hydrophilic pore
- target of many toxins and medicines
- mutation in one of these channels causes insensitivity to pain
- they switch from an open to a closed position
39
how do carrier proteins work
- bind directly to the solute
- participate in passive transport down a conc grad
- also do active transport using energy against a conc grad
40
what is symport
same as cotransport
both in
41
what is antiport
one goes in and one goes
42
advantages of carrier proteins
enhances the rate of transport at low solute concs
43
why have a nucleus
the nucleus separates the process of transcription and translation
44
why separate translation and transcription
- checks the quality of mRNA
- allows processing of mRNA
- allows repeated use of one mRNA increasing efficiency
- allows separation of transcription and translation rates
45
46
how is DNA packaged in the nucleus
- DNA is packaged into chromosomes
- DNA is wound around histones
- chromosomes occupy specific teritories within the nucleus but these can change
47
why can chromosomal territories change
- development and differentiation
- genetic mutations- response to DNA damage
- chromosomal rearrangements
- infectious disease
48
domains of the nucleus and what they do
Nucleolus - ribosome synthesis
speckles - pre-mRNA processing
cajal bodies - splicing
PML bodies - storage depot
49
how is the nucleus dynamic
the location of a gene within the nucleus depends on its transcriptional status
50
nuclear envelope
double unit membrane perforated with pores and supported by the lamina
asymety is maintained by teh lamina
51
what are lamins used for
- structural integrity
- gene regulation
- in plants its important for nuclear architecture
52
how does material enter and exit the nucleus
nuclear pores
they can open up to 26nm and particles with a MW>50,000 cant enter by simple diffusion
53
what is needed for proteins to enter the nucleus
- energy
- signals
54
structure and function of mitochondria
Outer Membrane
- contains porins that allow transport of molecules
Intermembrane Space
- H is pumped here to create a proton grad for oxidative phosphorylation
Inner Membrane
- proteins for ATP synthesis and transport proteins for matrix
Matrix
- contains enzymes for krebs cycle
55
where do mitochondria move to
subcellular locations where there is high energy demand
56
chloroplast structure and function
Outer Membrane
- contains porins that allow transport of molecules
Intermembrane space
Inner Membrane
Stroma
- contains DNA, ribosomes and RNA
Thylakoids
- photosynthesis
57
similarities of chloroplasts and mitochondria
- thylakoid membrane is the equivalent of the inner membrane
- both contain their own genetic material
- circular chromosomes
58
how are proteins delivered to mitochondria
- proteins to be translocated have a signal sequence
- mitochondrial proteins have a signal that directs them to the right location
- mitochondrial proteins are fully synthesised in the cytoplasm
- they associate with protein chaperones to prevent them from folding
- they are post-translationally translocated
59
what to the complexes that translocate proteins consist of
- receptor subunits that recognise the signal
- translocation channels that allow the protein to cross the membrane
60
how is translocation across the OMM and IMM coupled
- signal binds to receptor
- protein is translocated into intermembrane space
- recognised by receptor on IMM
- signal is cleaved off
61
why does mitochondrial translocation require energy
- to dissociate chaperones from polypeptide
- signal sequence is +vely charged
- electrochemical gradient drives ATP production and +ve charged signals through IMM
62
how are proteins delivered to chloroplasts
- similar to mitochondria
- different protein complexes with similar funcs
- signal, chaperones, receptor/channel complexes
63
how are proteins inserted to bacterial membranes
similar principles apply to insertion into bacterial and mitochondrial outer membranes
64
what is the ER
a reticulum that can spread throughout the cell
it is dynamic
65
what is the ER connected to
nuclear envelope
66
structure of ER
consists of two domains SER, RER
ER forms hollow tubes and flattened sacs, the chambers are called cisternae
67
ER functions
1. quality control of membranes and proteins
2. synthesis
3. storage
4. detoxification
68
what does the SER do
synthesise phospholipids and cholesterol
synthesise steroid hormones
synthesis and storage of glycerides
storage of calcium
69
how are membrane proteins translocated to the ER
co-translationally
as they are being translated, they are translocated across the ER membrane
70
how does the ER lumen help fold newly made proteins
it is rich in chaperones
71
principles of vehicular transport
vesicles bud from a donor membrane and are targeted to an acceptor
anything in the lumen or membrane of the vesicle will be transported to the new compartment
72
what does vesicle coating help with
coats provide structural stability to the forming vesicle and help with cargo selection
different types of coats act at individual transport steps
73
how do vesicles reach the right compartment
SNARE proteins act as address labels
v-SNARES found in the vesicle
t-SNARES found in the target membrane
74
structure of the Golgi
composed of flattened discs called cisternae
75
function of the golgi
- cisternae communicate with ER and cell membrane using vesicles and tubules
- modification and packaging of secreted proteins
- delivery of material to other organelles, including the plasma membrane
76
what does the trans golgi network do
it is a major sorting station for newly made proteins
from the golgi to the endocytic pathway
molecules can be delivered to the secretory vesicles which fuse with the plasma membrane when cells are stimulated by a signal
77
what is autophagy and what is it used for
It is 'eating of self'
-for normal homeostasis
- is a stress response
- for cellular remodelling
- protects against pathogens
78
what is endocytosis
large macromolecules enter cells by endocytosis. It can carry
- nutrients
- signals
- antibodies
- pathogens
- membrane
79
fates of endocytosed material
1. recycling to the cell surface
2. transcytosis
3. targeting to lysosome
80
what does multiple entrances allow
material to be internalised
it is specialised for different cargoes
81
what is phagocytosis
uptake of large particles
- pathogens, apoptotic cells
- bacteria are often coated by antibodies: opsonisation
82
what is macropinocytosis
1. cells form actin driven ruffles that can fuse to form macropinosomes
2. mechanically similar to phagocytosis
3. non selective uptake of extracellular material into the macropinosome
4. used by cancer cells to uptake nutrients
83
clathrin mediated endocytosis pathway
1. LDL binds with a high affinity to receptors on cell surface membrane
2. endocytosis occurs with a clathrin coated vesicle
3. vesucle is uncoated and fuses with endosome
4. transport vesicle buds off containing the receptor
5. LDL receptor returns to plasma membrane
84
clathrin coated pits and vesicles apperarance
a regular lattice
85
how do vesicles pinch off from the cell surface
using dynamin
86
cell pH gradient
a pH graidient is essential for endocytic trafficing
7.2 - plasma membrane
6.5 - early endosome
5.5 - late endosome
4.5 lysosome
87
how is cargo targeted for degradation sorted
in intraluminal vesicles (ILVs)
88
what are lysosomes
degradative compartments
optimal pH is 4.5 for proteases and lipases inside
89
BBoL
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