Golgi, vesicle transport, cytoskeleton

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What is the golgi apparatus made up of?
ER, Cis golgi network, Trans golgi network, secretory vesicles and plasma membrane or other organelles.
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What is the cis golgi network made up of?
cis cisterna, medial cisterna and trans cisterna
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What part lies nearest the ER and is at the site where vesicles from the ER dock?
The cis
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What part is the site where vesicles depart from?
The trans face
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What is the golgi appartaus?
Stack of flattened membrane bound compartments (cisternae)
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What are the functions of the golgi apparatus?
Proteins for secretions are sorted, modified and dispatched from the golgi. Also a major iste of carbohydrate synthesis in the form of glycoproteins and proteoglycans.
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What cells are the golgi prominent in?
Secretory cells like intestinal goblet cells.
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What chaperone recognises a single glucose of incompletely folded proteins?
calnexin
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What does calnexin do?
It prevents the incompletely folded proteins exporting to the golgi.
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How many glucose molecules are removed during the initial phases of folding?
2 of the 3 glucose molecules are removed
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What happens after the final glucose is removed for the dispatch from ER to golgi?
-If it is partially folded, glucosyl transferase adds another glucose and tries again.
-Misfolded proteins are chaperoned back to ER protein translocator and sent to cytoplasm to be degraded
-Correctly folded proteins go on to be exported to the golgi
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How do fully folded proteins exit the ER?
-soluble proteins are bound to transmembranous receptors
-Protein then need exit signals for efficient report -Most exit signals are known but not all eg. ERG1C53 receptor binds to mannose to factors V and VIII- blood clotting
- Coat protein COPII intera
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How are transport vesicles moved?
Once COPII coated vesicles bud off from ER, rapidly shed coat
-undergo homotypic fusion- like joining like
-vesicular tubular clusters moves along microtubules- delievers content to golgi
-once cargo reaches golgi, released from receptor and is mediated b
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How are proteins transported back to the ER?
- proteins from er budding retrieved from VTC (recycled)
-proteins escaped from ER by mistake need retrieving -COPI coated vesicles bud from VTC/golgi-uncoated, transported back to ER
-Resident ER membrane proteins=cytosolic sequence interacts directly wi
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What happens in the ER in oligosaccharide processing?
n-linked glycosylation
folding and glucose trimming
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What happens in the cis cisterna in oligosaccharide processing?
Mannose trimmed
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What happens in the medial cisterna in oligosaccharide processing?
Mannose trimmed
N-acteyl glucosamine added
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What happens in the Trans cisterna in oligosaccharide processing?
addition of galactose and n-acteylneuraminic acid
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How is oligosaccharide processing ordered?
sugars are removed and added in turn, each step relies on previous one
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What is another type of modification in the golgi?
o-linked glycosylation in the cisternae, Phosphorylation of oligosaccharides on lysosomal proteins in the CGN, Sulphation of tryosine and carbs in TGN.
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What is o linked glycosylation?
addition of sugars to the OH group of SER, THR or hydroxylysine and is catalysed by glycosyl transferases
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What is usually added first in o linked glycosylation?
n-acetyl galactosamine
Additional residues ranging from a few to over 10 are then added
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What is the point of glycosylation?
As a protein marker, as a protector, as a cellular marker, regulatory roles
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What do protein markers do in glycosylation?
for complete folding, targets transport between er and golgi, sorting in the golgi
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How does glycosylation acts as a protector?
oligosaccharides prevents proteases approaching and digesting extracellular proteins
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What do cellular marker do in glycosylation?
oligosaccharides on cell surface proteins form part of the cell-cell recognition and adhesion mechanism.
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What regulatory roles does glycosylation do?
Cell surface signalling receptors may be glycosylated and alterations to their glycosylation pattern may affect activity in different cell types.
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How are molecules transported through and out the golgi?
-ER passes vesicles to VCT
-CGN occurs and phosphorylation of oligosaccharides on lysosomal proteins-sorting-Removal of mannose in cis cisterna then another in medial
-trans cis adds oligo and NANA
-TGN sulfation of tyrosines and carbs=sorting (secretory
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What is vesicular transport?
A vesicular transport protein is a membrane protein that facilitates movement of specific molecules across vesicles membrane
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What are the principles of vesicular transport?
-Membrane bound vesicles carry cargo in lumen from donor to a recipient compartment-TGN.-vesicles generated in donor. Recipient is where they receive.
-Vesicles bud from coated regions of donor
-coat is discarded, allows membranes of vesicle and recipient
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What is clathrin?
coats the vesicles which transport molecules between the golgi, lysosomes and plasma membrane
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What is COPI?
coats vesicles which transport molecules from golgi back to ER.
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What is COPII?
coats vesicles which transports molecules from er to golgi
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What does clathrin contain?
3 large and 3 small polypeptide chains which forms a three legged triskelion.
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What do the triskelions form?
forms a basket like convex framework of pentagons and hexagons on cytosolic side of membranes
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What is the role of clathrin in transport?
-Second protein complex, adaptin, needed to attach the clathrin coat to membrane
-adaptins interact with transmem proteins like TMreceptors-captures soluble cargo
-4 different adaptins, each recognise different set of cargo receptors
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What is dynamin?
Protein that binds in a ring to the neck of the budding vesicle. It pinches off the vesicle
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What does the pinching off of the vesicle cause?
causes the clathrin coat to be rapidly lost and recycled and naked vesicle is ready for transport
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Why do vesicles have surface markers?
to identify them according to origin and cargo
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What snare works with v-Snare?
t-snare is complementary to v-snare
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What helps give the vesicles information?
the v-snares
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What forms when the v and t snare meet?
they wrap around one another, forming a stable trans-snare complex.
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What does the trans snare complex do?
They lock the two membranes together- docking
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What are rab proteins?
they mark the vesicles
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What are rab proteins recognised by?
rab effector proteins.
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what can rab effector proteins do?
tether proteins on target membrane or it can help snares.
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What happens after the fusion of the snares?
for the snares to fuse, they need to be in close proximity so water is excluded. Formation of trans snare complex acts as a winch, using energy released as the helices wrap to fund water exclusion and membrane fusion
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What is an example of a type of pathway that is considered as exocytosis?
constitutive secretory pathway
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What does the constitutive secretory pathway do?
-secretes components of the extracellular matrix
-delivery of new plasma membrane proteins and lipids to cell surface
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What causes proteins to be transported by the default pathway?
any protein that isn't returned to the ER, retained in the golgi lumen, directed to lysosomes, regulated secretion
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What happens after vesicles bud from the trans golgi network?
they are targeted to the plasma membrane and they fuse with is immediately.
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What does the regulated secretory pathway do?
they allow a concentration of secretory proteins so that is a buildup of the protein within a particular compartment increasing its conc in that part of the cell
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What do secretory granules do?
They are cells that concentrate and store secretory products in special vesicles
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What do secretory proteins do in the acidic conditions of the golgi?
They aggregate (accumulate) then leave the TGN as loosely bound immature secretory vesicles
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What increases as the vesicles mature?
The concentration of secretory proteins
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Why does the conc increase as vesicles mature?
retrieval of the membrane back to the golgi, increased acidity in the maturing vesicle lumen, causing tighter aggregation
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What does the second regulatory pathway do?
allows further processing of secretory proteins.
-allows release in response to a trigger
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What is further processed in pathway 2 and what is it processed by?
Secretory vesicle proteins may be further processed by proteolysis like peptide hormones, neuropeptides and secretory hydrolytic enzymes
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What does further processing of secretory vesicle proteins allow?
enables short (5AA) peptides to be made-neuropeptide
-Protects cell from own hydrolytic enzymes
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When does fusion occur for some cells?
when a specific trigger is shown. Some cells vesicles dock with plasma membrane
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What is an example of pathway 2?
digestive enzymes are secreted in response to food in the gut
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What is endocytosis?
following the release to secretory proteins to extracellular space, there is a transient increase in the surface area of the plasma membrane. The extra components must be removed and recycled.
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What is balanced in endocytosis?
secretion and internalisation
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What causes the balance?
the endocytotic-exocytotic cycle
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What are the types of endocytosis?
pinocytosis, receptor mediated endocytosis, phagocytosis
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What is pinocytosis?
takes up things on plasma membrane- controlled by clathrin-invaginates the membrane to make a pit for cargo to be taken in opposite direction. Then forms vesicles and lose their coat and fuses with early endosomes- extracellular constituents are delivered
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What is receptor mediated endocytosis?
LDL containg cholesterol interact with LDL receptors on animal cells
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What is phagocytosis?
occurs in white blood cells/lymphocytes. Takes dead cells and large cells like bacteria- stimulates cells to extend pseudopods- used to engulf particle. Results in large endocytic vesicle- phagosome- lysosomes fuse with it and destroys it.
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What are lysosomes?
They are the major site of intracellular digestion and are membrane bound compartments. The enzymes are acid hydrolases
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What do lysosomes contain?
40 types of hydrolytic enzymes like proteases, nucleases, glycosidases, lipases
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What are the enzymes in the lysosome maintained at?
at pH5 by a hydrogen ion pump in lysosomal membrane.
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How are the cells protected from its own digestive enzymes?
-lysosomal membrane keeps the enzymes out the cytosol
- acid hydrolases dont work at pH7
- lysosomal hydrolases are protected from each other by glycosylation
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What do they use energy in the form of?
Form of ATP for the pump
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What are the functions of lysosomes?
-hydrolytic enzymes transported from ER where they are synthesised
-macromolecules targeted for digestion come from variety of sources
-digestion products (sugars, aa, nucleotides) transported out lysosome by transporters in membrane
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How does transport to the lysosome happen part 1?
-Signal peptide directs synthesis of lysosomal proteins into ER- n linked glycosylated
-CGN-signal patch directs phosphorylation of terminal mannose on n linked oligosaccharide
-mannose6phosphate=signal for lysosomal delivery -TGN M6P receptors in membran
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How does transport to the lysosomes happen part 2?
-Transport vesicles fuse with late endosome acidic pH causes m6p receptor to dissociate from m6p labelled hydrolase, into lumen.
-signal in cytoplasmic tail of m6pr targets it into transport vesicle-taken back to TGN
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How does transport to the lysosomes happen part 3?
-late endosome, phosphate is removed from mannose sugars on hydrolases to ensure not returned back to the golgi
-As h pump lowers the pH, acid hydrolases digest macromolecules from early endosomes and then endosomes matures into lysosomes.
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What are the usual routes out of the lysosomal membrane?
-passive diffusion, small hydrophobic molecules pass out
-membrane transporters, digestion products trasnported out by transporters
-secretory lysosomes, lysosomal secretion allows cells to eliminate indigestible debris- usually used under stress
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What happens when endocytosed molecules fuse with early endosomes?
they are put in the sorting site then:
-either recycled- vesicles return to PM
-transcytosis-vesicles return to different parts of PM, transports material across cell
-degradation- cargoes sent to late endosomes-matures into lysosomes, macromolecules degr
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What are peroxisomes?
bags of oxidative enzymes which may have crystalline core. They are the sites of oxidative reactions and generate hydrogen peroxide
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Where are peroxisomal proteins produced?
in the cytoplasm and are targeted to peroxisomes by a c terminal signal -ser-lys-leu. Proteins targeted to peroxisomes by a signal which means they are fully folded when imported into peroxisome.
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What are the 23 distinct proteins called?
peroxins- involved in soluble receptor proteins in cytosol and docking proteins on cytosolic surface
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How are new peroxisomes formed?
by fission once the parent reaches a certain size. fusion occurs to reduce numbers
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What is the structure of the mitochondria?
-matrix
-inner membrane
-outer membrane
-intermembrane space
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What are the functions of mitochondria?
-Powerhouse of the cell-generates energy for the cell
-most foods broken down in cytoplasm to simple constitutes which then transported to mitochondria
-once in, further oxidation occurs-produces 02 and h2o releasing energy as ATP
-located close to sites
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What is an example that has high amounts of mitochondria?
sperm as they need energy to move
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What are the resulting molecules of the hydrolysis of ATP?
ADP or AMP and inorganic phosphate. To regenerate ATP, energy is required from another source
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In the cytoplasm what do sugars and fats turn into?
sugars turn into pyruvate and fats turn into fatty acids and glycerol
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in peroxisomes, what do fatty acids turn into?
acetyl coA
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What are the compounds above transported into?
mitochondrial matrix
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What is first converted in the mitochondrial matrix?
Fats are first converted into acetyl coA and then fully oxidised by krebs cycle
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What is generated after the conversion of acetyl coA?
co2, some energy (GTP) and NADH and FADH2
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What is oxidative phosphorylation also referred to as?
Electron transport chain
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What happens in oxidative phosphorylation part 1?
-NADH and FADH2 generated in cytoplasm by krebs cycle and donates electrons so nadh=nad+ h+2e- and fadh2=fad+2h+2e-
-FADH and NADH arrive at inner membrane and donates electrons to protein
-NADH dehydrogenase complex accepts 2 electrons in complex
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What happens in oxidative phosphorylation part 3?
High energy electrons pass down 3 complexes in inner membrane-ubiquinone is the carrier and transfers it to next protein in sequence=cytochrome b-c1 complex
-next transfer is done by cytochrome 2 (carrier) to next protein- finally ends up with 02 as fina
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What happens in the electrochemical gradient?
-transfer from lower to higher electron affinity=energetically favourable
-energy released used to pump h+ into intermembrane space
-pumping h+=electrochemical gradient across inner mitochondrial membrane
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What is proton motive force?
-ATP synthase in inner membrane forms hydrophillic pathway-h+flow down gradients -h+flow causes rotation of transmembranous rotor domain-attached stalk rotates like a water wheel- lollipop shaped enzymatic head-held still by arm attached to membrane
-mec
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How much energy is released from proton motive force?
2.5atp from NADH and 1.5atp from FADH2
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What does the outer membrane of the mitochondria have?
Large pores which are made out of porins- makes it permeable to molecules
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What are co-transported in the electrochemical gradient?
pyruvate and inorganic phosphate transport is driven by h+ gradient- co transport in same direction (symport)
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What is ATP and ADP driven by in the transport in/out of mitochondria?
by a voltage gradient:
-ATP has a 4- charge
-ADP has a 3- charge
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What does the co transport of ATP out and ADP in to the mitochondrial matrix cause?
causes a net change of 1- in the innermembrane space which has a positive electrical charge, this makes the reaction favourable
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Where are most proteins made for the mitochondria encoded?
in the nucleus and are produced by cytosolic ribosomes
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What happens to mitochondrial proteins in the cytoplasm?
they are chaperoned, keeping them unfolded while their n terminal signal peptides target them to the mitochondria
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What do proteins pass through to get to the mitochondria?
TOM (translocases of the outer mitochondrial membrane) and TIM (translocases of the inner mitochondrial membrane). Proteins can either enter one (tom) or both to become localised to either enter inter membrane space or the matrix. Stop transfer signals en
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Where does transcription and translation occur in the mitochondria?
in the matrix/stroma
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What were mitochondria and chloroplasts most likely as?
originally as bacteria which entered a symbiotic relationship with a primordial nucleated cell. They are not synthesised from scratch so must've grew and divided.
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What is the cytoskeleton?
It is the cells skeleton and is a filamentous structure which is found through out the cytoplasm and in the nucleus
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What is the cytoskeleton formed from?
protein monomers which assemble into repeat structures
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Why is the cytoskeleton classed as dynamic?
is assembles and disassembles to suit the cells needs
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What are the filaments that make up the cytoskeleton?
actin filaments
microtubules
intermediate filaments
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What drug affects the assembly and disassembly of the cytoskeleton?
taxol
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What are actin filaments?
globular protein actin which assembles into 2 stranded helical polymers. Lines up to form bundles/ 2d networks/ 3d gels.
-concentrated beneath the cortex
roles in cell shape and motility
-5-9nm diameter
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what are microtubules?
made from globular protein tubulin (a and b subunits)
-dimerize to form hollow tubules and much more rigid than actin
-long and straight, 25nm
one end attached to MTOC other end grows and shrinks.
-roles in positioning organelles, mitosis and intracellul
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What are intermediate filaments?
made of various intermediate filament proteins which are filamentous.
-roles in mechanical support of cell structures
-10nm diameter
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How are cytoskeletons formed?
-monomers form end to end and side to side interactions- noncovalent rapid assembly/dis no bonds to break compared to DNA,RNA,peptides
-rate of disassembly is constant
-
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What happens when there is critical concentration of monomers?
addition and removal happens at the same rate. When cytoskeletal elements are at equilibrium phase there is no net growth but still gradually turn out-forward and backward reaction occurring at the same time
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What can effect processes allowing cell control?
auxiliary proteins
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What do both actin filaments and microtubules have?
both have fast positive and slow negative growing ends
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When does a conformation change happen?
when subunits attach to the + end. This readies them for the next monomer and gives the + end a higher affinity for monomers than the - end.
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What happens when nucleotides are hydrolysed?
-Actin monomers carry an ATP molecules
-actin-atp has a high affinity for actin polymer
-shortly after addition to the filament the ATP is hydrolysed to ADP.
-Actin-adp has a lower affinity for the polymer
tubulin heterodimers carry a GTP molecule
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Accessory proteins may be useful to the formation of the cytoskeleton. What do they do?
-Dynamic (constantly changing) control of filaments: nucleation, assembly, disassembly
-Link other cellular components to the cytoskeleton
-motor proteins
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What is nucleation?
auxillary proteins may act as an initiator for a new filament
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What is assembly?
proteins which bind monomers change their relative concentration, affecting addition rates
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What is disassembly?
Proteins can bind and either stabilise or destabilise filaments
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How do accessory proteins link other cellular components to the cytoskeleton?
Actin filaments often emanate from the cell membrane where they are attached by a variety of protein complexes which can also interact with the extracellular matrix and other cells
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What are motor proteins?
Move organelles along filaments against each other
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What are myosins?
They are motor proteins that are associated with actin filaments
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How many myosin genes do humans have?
40
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What myosin makes muscles contract?
myosin II
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How is a long tail domain formed?
two myosin II heavy chains coil
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How are large bipolar thick filaments formed?
tail domains interact with other myosin pairs to form them with hundreds of myosin heads. The globular head region contains the force generating machinery.
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How do myosins work as motors?
-attached: myosin head bound to actin filament
-release: myosin binds to ATP and releases actin
cocked: ATP hydrolysis and conformation change
-grip- reattachment to actin and phosphate release
-pull: conformation change and ADP release.
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How does muscles contract?
Sarcomeres are arranged in long repeating chains-myofibrils.
-Each sarcomhere contains partially overlapping thin+thick actin filaments
-Motor action myosin pulls actin filaments-slides over one another
-Neither filament changes size but sarcomeres shorte
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how do kinesins as motors work?
- leading head binds to tubulin. Trailing head binds to ADP
-leading head binds ATP causing a conformation change which throws trailing head forward
-trailing head binds tubulin
-trailing head releases ADP while leading head hydrolyses ATP.
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What does dynein do?
retains golgi close to the centrosome
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What do kinesins do?
disperses ER to cell periphery, which walki it to the + ends along microtubules.
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What are large complexes needed for?
to attach dynein to membrane bound organelles and vesicles
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What is cell crawling?
involves rearrangement of the actin cytoskeleton.
-protrusion: actin fibres form at leading edge
-attachment: actin cytoskeleton attaches to the surface across the cell membrane at focal points
- traction: myosins pull the trailing cytoplasm forward
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What is an example of cell crawling?
-flagella and cilia-sperm
tubular structures composed of microtubules. Motor activity of ciliary dynein causes the microtubules to slide across one another, causing flagellum to undulate and propel the cell forward.
-microvilli-in the gut epithelium
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What does microvilli in the gut do in terms of cell motility?
Bundles of actin filaments extend to the tip
myosins attached to the cell membrane walk along the actin filaments causing the microvillus to wave.
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Card 2

Front

What is the cis golgi network made up of?

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cis cisterna, medial cisterna and trans cisterna

Card 3

Front

What part lies nearest the ER and is at the site where vesicles from the ER dock?

Back

Preview of the front of card 3

Card 4

Front

What part is the site where vesicles depart from?

Back

Preview of the front of card 4

Card 5

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What is the golgi appartaus?

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