cytoskeleton and light microscopy

fluorescence

ability of a substance to absorb light of one wavelength and emit light of a lower wavelenghth, difference is called the stroke shift

fluorescence proteins

proteins with genetic mutations that allow them to be fluorescent. these can be genetically encoded to be added to pre-existing proteins for the purpose of research.

flourescence microscopes

first filter out everything but blue light, when is then reflected downwards through a wave splitting mirror. then it goes through an objective lenses, gets emitted as a new wavelength from the object, goes through the beam splitting mirror, then through a final light filter before it reaches the camera. 

fixation

the use of killed cells to study biology, after the cells die you permeabilize(make the membrane temporarily Porus) and then stain them

immunostaining

the use of antibodies to mark proteins which then attracts markers that let you track your target molecule

microinjection

the use of a micropipette to inject substance into a cell

electroporation

cells are briefly shocked opening the membrane allowing substances to enter

lipofection

vesicles containing the substrate are fused with your target cell causing the substrate to enter the cell

gene gun

your target dna is attached to gold particles which are shot at the cell inserting them

affinity chromatography

cell lysate containing your protein of interest is poured onto a burrette containing beads with a ligand which will attach to your protein. after this the burrete is washed with a buffer that removes everything except the beads ligands and target proteins. after this an elution buffer is passed through which removes the target proteins from the ligands

production of antibodies

a mouse is injected with an antigen, this causes its spleen to produce immune cells that have the corresponding antibodies. these cells are then cured with myeloma cells and then you isolate the cells containing the antibodies you want from this.

actin

polarized chain of proteins that plays an important role in cells as part of the cytoskeleton and a pathway for motor proteins

G-actin

individual actin subunits

f-actin

the chain of g-actins

actin polymerization

at the positive end g-actin is polymerized, and at the minus end it is removed. it is attached using phosphorylation and after it is attached it is hydrolyzed to adp+p when the phosphate gets removed it the gtp detaches

treadmilling

when the rate of g-actin addition to the actin equasl the rate of subtraction

pyrene actin essay

graph showing the growth of actin over time, has three phases, the lag phase growth phase and equilibrium phase

nucleation

the formation of the first chunk of actin which is used to build the f-actin

lag phase

part of the pyrene actin essay, corresponds to the amount of time it takes for nucleation to happen

actin polymerization at each end

at the +(barbed end) actin polymerization is very fast causing it go grow at the - (pointed end) actin polymerization is slower leading to very slow or negative growth

gelsolin

protein that cleaves actin increasing the rate of growth by increading the number of + ends

Arp2/3 complex

eliminates the need for a nucleation step by acing as the minus end of the actin filiment

plus end caping protein

protein that binds to the plus end of actin. stops growth at the plus end results in net slower growth or shrinkage.

myosin

motor proteins with a tail and two heads that walk along actin filaments. have a heavy chain(the tail) and light chain(the hinge region)

myosin regulation

myosin activity can be regulated by sites on the light and heavy chains which can be phosphorlated

myosin movement cycle

starts at 90 degree angle to the actin then atp binds and hydrolysis’s causing it to release. after that the myosin swings forward and reattaches releasing the phosphate. after that it swings backwards releasing the adp

myosin variation

many different types of myosin. function is dependent on their structure but the basic mechanism is always the same.

myosin 1

has only one head often used to move f-actin or vesicles

myosin 2

myosin with two heads forms multiple oligomers of dimers in order to rearrange f-actin

actomyosin regulation

regulated by calcium levels. when present calcium binds to tropomyosin pulling the troponin away from the binding sites on the actin letting myosin move it

microtobules

polarized cylinders made up of thirteen protofilaments part of the cytoskeleton, serve a multitude of purposes in cells

tubulin

heterodimer that makes up the protofilaments in microtubules

minus ends of microtubules

often embed in in centrosomes. 

microtubule synthesis

starts from a base of gamma tubulin alpha tubulin and other accessory proteins

microtubule

tubulin with gtp attaches will bind to form microtubules, when the gtp gets dephosphorylated to gdp the microtubule weakens and comes apart.

dynamic instability

property of microtubules to rapidly shrink and retract. occurs when the plus end of the microtubule starts to peel away causing a rapid break down of the microtubule

stathmin

protein which binds to free tubilin making it unable to attach to the microtubules. after this happens the rate of dephosphorylation catches up with the rate of hydrolysis causing the microtubules to retract. 

motor protein regulation of microtubules

motor proteins can bind to the end of microtubules either stabilizing or destabilizing them, causing them to either grow or shrink

microtubules and cell shape

the movement of microtubules can alter and shift cell shape

kinesin

microtubule motor proteins that move towards the plus end

dynein

microtubule motors that move towards the minus end

microtubule motor proteins

two headed proteins that walk along the microtubules transporting vesicles and other cargo

intermediate filaments

part of the cytoskeleton. exist in long chains which are weaved into a rope like structure. not polarized exist in many different forms like keratin and neurofilaments

intermediete filiment assembly

intermediate filaments start as a monomer then coil into a dimer which gets stager with another dimer then two of these tetramers get packed end to end. finally eight of these tetramer chains get twisted into a rope

filament system interactions

the different types of filaments can interact with each other. often linked by proteins like plectin which links all three types of filaments

role of intermediate filaments

intermediate filaments allow large groups of cells to withstand stretching’s and pulling without rupturing.

Rho GTPases

tightly regulated proteins which impact the function of the cytoskeleton

cytoskeleton regulation

mostly done by g-proteins which control the most important effectors