Midterm 2 (Lectures 8-9) Flashcards
properties of microtubules
hollow tube with a wall consisting of typically 13 protofilaments. has + and - ends (polarity)
properties of microfilaments
two intertwined chains of F-actin. has + and - ends (polarity)
properties of intermediate filaments
eight protofilaments joined end to end with staggered overlap. no known polarity
what are the subunits incorporated into the polymer of microtubules
GTP-alpha beta-tubulin heterodimer
cell polarity
spatial differences in shape, structure, and function within a cell
kinesin
anterograde MT motor, moves from negative end to positive end
dynein
retrograde MT motor, moves from positive to negative end
what are microtubules
straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments
basic subunit of a protofilament
a heterodimer of tubulin, one alpha-tubulin and one beta-tubulin (globular proteins) which bind non-covalently to form aB-heterodimer
MAPs
microtubule associated proteins. heterogeneous collection of proteins with one domain attached to the side of a microtubule and another domain that projects outwards as a tail
structure of microtubules
a dimer of alpha tubulin and beta tubulin noncovalently bound. they are always found together in the cell. a-tubulin has GTP and B-tubulin has GDP. each monomer has a GTP binding site. Taxol is on B-tubulin
GTP in a monomer
is physically trapped at the dimer interface. it is never hydrolyzed or exchanged
in the beta-tubulin monomer:
the nucleotide can be in either GDP or GTP form and is both hydrolyzable and exchangeable
draw the structure of a MT
(-) end has a-tubulin with GTP, (+) end has B-tubulin with GDP
molecular motors
move unidirectionally along their cytoskeletal track in a stepwise manner
how do kinesin, dynein, and myosin move
kinesin, dynein move along microtubule track, myosin moves along microfilament tracks
kinesins and cytoplasmic dynein moves:
similar materials in opposite directions over the same railway network. Organelles may bind kinesin and dynein simultaneously. Dynein and kinase can be found on the same microtubule.
how do vesicles know what membranes to fuse with?
SNAPS and SNARE
nucleus
- nuclear envelope
- mRNA, mRNP and proteins need to exit and enter
- everything exits and enters via nuclear pores
smooth endoplasmic reticulum is involved in:
- drug detoxification
- carbohydrate metabolism
- calcium storage
- steroid biosynthesis
golgi apparatus function
site of post-translational processing of lipids and proteins synthesized in the ER and sorting for transport to other sites in the cell
mitochondria
- not part of endomembrane system but site of ATP formation
peroxisomes
breakdown fatty acids two carbons at a time into acetyl CoA, found in all animal cells except RBCs
lysosomes
contain ~40 types of hydrolytic enzymes, all of which are acid hydrolyses. also contains transport proteins that carry the products of macromolecule digestion to the cytosol
endomembrane system - biosynthetic pathway
proteins are synthesized in the ER, modified at the golgi complex and transported to various destinations
endomembrane system - secretory pathway
proteins synthesized in the ER are secreted from the cell
endomembrane system - secretory pathway - constitutive secretion:
materials are transported in secretory vesicles discharged in a continual manner
endomembrane system - secretory pathway - regulated response:
materials are stored in vesicles and discharged in response to a stimulus
subcellular fractionation
homogenization followed by centrifugation
differential centrifugation
separation of cell organelles or other particles of different size/density by their different rates of sedimentation in a centrifugal field
density gradient (rate zonal) centrifugation
a sample (often isolated first by differential centrifugation) is further separated/fractionated by layering the sample on top of a sucrose gradient
what happens when you centrifuge:
a) at 500 x g for 10 minutes
b) at 10,000 x g for 20 minutes
c) at 100,000 x g for 1 hour
a) supernatant - pellet: nuclear fraction
b) pellet: mitochondrial fractions
c) cytosol (soluble proteins) - pellet: microsomal fraction
what is the purpose of a microscope
detect, magnify, resolve
magnification
enlarges but does not distinguish between two points
resolution
the minimum distance that can distinguish between 2 points
decreasing wavelength -
increases resolution
light microscopy
- live cells and tissues lack compounds that absorb light and are therefore nearly invisible in a light microscope
- unstained
4 types: - bright field
- phase contrast (improves contrast)
- Nomarski optics (uses special prism to split illuminating light beam into 2 separate rays)
- dark field (excludes unscattered beam from the image)
microscopy: fixation
preserves cells; prevents decay/degradation, uses heat, formalin, glutaraldehyde
microscopy: staining
- doesn’t increase resolution, just enhances visualization
- adds colour for contrast
- basic dyes bind to (-) charges on proteins and nucleic acids, acidic dyes bind to + charges on proteins and phospholipids
microscopy: fluorescent dyes
- used to increase resolution and localize and quantify specific molecules in fixed and living cells
- light is absorbed at one wavelength and emitted at a longer wavelength
- used for staining cells
using flurochromes (fluorescent dyes)
- labels specific proteins by attaching the dyes to an antibody molecule
- antibody is attached to a protein, the fluorescent marker attaches to the antibody
direct immunofluorescence
- specific antibodies against antigen
- antibodies labeled with fluorescent dye
- allow antibodies to bind to antigen
indirect immunofluorescence
- specific antibodies against antigen (primary antibody)
- allow antibodies to bind to antigen
- add labeled antibodies that bind to primary antibodies (secondary antibody)
trans golgi network
functions in sorting proteins either to the membrane or various intracellular destinations
medial cisternaie
where processing takes place
cis golgi network
functions to sort proteins in the ER or the next golgi station
glycosylation
oligosaccharide processing in the golgi complex
O-linked glycoproteins
not as abundant as N-linked. abundant in extracellular matrix
glycosylation of ceramide:
occurs in the golgi apparatus
ceramide:
sphingosine backbone and a fatty acid tail
vesicular transport model
vesicle budding from cis to trans golgi via antegrade transport along with retrograde transport
cisternal maturation
cis golgi network matures into medial golgi and then trans golgi network but also retrograde transport
materials are carried between compartments using
coated vesicles
protein coats have two distinct functions
- cause the membrane to curve and form a vesicle
- select the components to be carried by a vesicle
KDEL sequence
is both necessary and sufficient to result in a protein containing this sequence to be located in the ER
- KDEL sequence binds to a KEDL receptor - a transmembrane protein
if you added the KDEL sequence to a protein that is normally secreted - what will happen to it?
won’t be secreted, will come back to the ER
T-SNARE and V-SNARE
T - for target membrane
V - for vesicle membrane
clatherin coats
- involved in trans-golgi network to late endosomes/lysosomes
- plasma membrane to early endosomes
clatherin:
major protein component of clathrin coated vesicles
Triskelion
a clathrin molecule
- each clathrin molecule consists of three large and three small polypeptide chain
sorting and transport of lysosomal enzymes
- utilizes clathrin-coated vesicles
- lysosomal proteins are tagged in the cis golgi with phosphorylated mannose residues
- tagged lysosomal enzymes are recognized and captured by mannose 6-phosphate receptors (MPRs), which are bound by coat proteins
early endosomes:
are generated by clathrin coated receptor mediated endocytosis
autophagosome
a double-membrane sequestering vesicle that forms when a double membrane structure envelops an organelle
autophagosome fuses with a lysosome and the enclosed contents are degraded in a:
autolysosome
lipofuscin granule:
when the contents of a residual body are retained inside the cell
exocytosis
discharge of a secretory vesicle or granule after fusion with plasma membrane. process triggered by an increase in Ca2+
steps of exocytosis:
- approach of secretory vesicle to plasma membrane
- fusion of membranes
- rupture of plasma membrane
- discharge of vesicle contents to the outside of the cell
endocytosis:
a process by which the cell internalizes cell-surface receptors and bound extracellular ligands
two types of endocytosis
- bulk phase - non specific uptake of extracellular fluids
- receptor-mediated or clathrin-mediated - brings about the uptake of specific ligands following their binding to receptors on the external surface of the plasma membrane
steps of endocytosis
- membrane invaginates, forming a pocket containing macromolecules or other materials from the exterior of the cell
- pocket begins to pinch off, enclosing the extracellular material
- membrane closes around the invaginated material, forming a vesicle
- vesicle separates from the plasma membrane, carrying material from the exterior within a membrane derived from the plasma membrane
receptor mediated endocytosis
RME provides a selective and efficient uptake of macromolecules. receptors are concentrated in coated pits at 10-20 times their level in the remainder of the plasma membrane. substances that enter the cell through clathrin-mediated RME become bound to coated pits on the plasma membrane
low density lipoproteins
- complex of cholesterol and proteins
- LDL receptors are transported to the plasma membrane and bound to a coated pit
- LDLs are taken up by RME and taken to the lysosomes, releasing the cholesterol for use by the cells
transcytosis
a combination of endocytosis and exocytosis
pulse-chase
follows the movements of newly synthesized molecules by observing a wave of radioactive material moving through the cytoplasmic organelles from one location to the next until the process is complete
what is the difference between RER and SER
RER has ribosomes bound to its cytosolic surface, whereas the SER lacks associated ribosomes
about ____ of proteins are synthesized in the RER
one third
what proteins are synthesized in the RER
- secreted proteins
- integral membrane proteins
- soluble proteins that reside in the ER, golgi, lysosomes, endosomes, vesicles and plant vacuoles
polypeptides synthesized on ‘free’ ribosomes in the cytosol include
- proteins destined to stay in the cytosol
- peripheral proteins of the cytosolic surface of membranes
- proteins that are transported to the nucleus and those incorporated into peroxisomes, chloroplasts, and mitochondria
co-translational translocation
transport of most secretory proteins into the ER lumen begins while the protein is still being synthesized on the ribosome. ER is all co-translational
post-translational translocation
if translocation takes place after the protein is synthesized
SRP:
- signal recognition particle
binds simultaneously and transiently to: - signal sequence in a nascent protein
- large ribosomal unit
SRP then binds (docks) to SRP receptor
single-spanning membrane proteins can have an orientation with:
their N-terminus facing either the cytosol or the lumen of the ER
chaperones and other ER proteins:
facilitate folding and assembly of proteins
only proteins that are properly folded can:
leave the ER.
what happens to proteins that are misfolded
- they are initially retained in the ER
- an accumulation of misfolded proteins functions as a signal for the cell to make more ER = unfolded protein response
- this response can only go on for so long. will eventually lead to apoptosis
dislocation or retrotranslocation
the movement of misfolded proteins from the ER to the cytosol
ERAD
- ER associated degradation proteins
- export improperly folded proteins from the ER to the cytosol for degradation by proteosome
phagocytosis
carried out by cells specialized for the uptake of relatively large particles. folds fuse to produce a vacuole (phagosome) that pinches off inwardly from the plasma membrane and fuses with a lysosome (phagolysosome)
posttranslational uptake of proteins by peroxisomes, mitochondria
- cannot fold
- unlike RER, which generally imports its proteins cotranslationally
- the proteins of these other organelles are imported posttranslationally, following their complete synthesis on free ribosomes in the cytosol
signal sequences
- direct proteins to the correct organelle
- is often removed following protein sorting
- are absolutely necessary for sorting
nucleoporins
- collective name for the group of proteins that make up the nuclear pore complex (NPC) (~30 different proteins)
- ions, small metabolites, and globular proteins can diffuse through the NPC
- larger proteins and protein complexes are selectively transported with the assistance of soluble transporter
GTPase
- very small proteins that bind guanine nucleotides
- hydrolyzes GTP that is bound to them
- have to hydrolyze GTP if you don’t want it on
GTPase switch proteins exist in two forms
- GTP bound - GTP is then hydrolyzed to GDP
- GDP bound - GTP is displaced by GDP
ran-GAP
GTPase Activating Protein
- found in the cytosol
ran-GEF
Guanine-nucleotide Exchange Factor
- found in the nucleus complexed to chromatin
importin
nuclear transport protein/receptor in cytosol that binds to cargo protein with an NLS and translocates to nucleus
exportin
nuclear transport protein/receptor in nucleus that binds to Ran-GTP which promotes binding to cargo protein
