Lecture 4 Flashcards
What microscope techniques can you NOT use to see cytoskeletons?
Light microscope cannot be used, it’s diameters range from 7nm to 25nm, and the resolution limit is about 200 nm and it limits the wavelength of visible light.
What microscope techniques can you use to see cytoskeleton?
1) Flurocent microscopy- it uses the same resolution as light microscopy however we can fluorescent label to detect specific proteins.
2) Transmission electron microscopy: uses beams of electron, very short wavelength and the resolution limit is about 1nm. It reveals detailed structure
What is immunofluorescene microscopy used for?
It is used to determine location of proteins within the cell
How does Immunofluroescenence microscopy work?
It is used to determine the location of a specific protein, and the cells are fixed (they are not imaging)
Primary antibody is used to bind to specific protein of interest and then secondary anti body binds to the primary antibody. (This is covalently tagged to the flurocent marker)
Then fluorescence microscope is used to excite fluorescent marker and visualize the light emitted.
What are the 3 types of filaments that form the cytoskeleton?
The smallest ones are Actin which are made of actin filaments
Next is Intermediate filaments which are made up of intermediate filament proteins and the largest are micro tumbles which are made up of tubulin
What sort of interactions are the microtubles filaments held together by?
Non covalent interactions
What are the four kinds of intermediate filaments
Cytoplasmic:
Keratin filaments (found in epithelial cells)
Vimentin and vimentin-related filaments ( found in connective tissue cells, muscles cells, and glial cells)
Neuro filaments (in nerve cells)
Nuclear:
Nuclear lamins (found in all animal cells)
What are the two types of intermediate filaments and what are their functions?
These are involved in structural support. And the cytoplasmic IFs: are in animal cells subjected to mechanical stress and they provide mechanical strength
Nuclear IF: they are in nuclear lamina and provide 2D mesh work. Formed by lamins in all animal cells (plants have different lamin-like proteins)
What are the structural characteristics of cytoplasmic intermediate filaments
These are conserved alpha helical central rod domain and has a different C and N terminals.
However, a monomer with polarity (different ends ) coils with another monomer making a dimer which is still polar. These are staggered anti parallel tetramer.
Then 8 tetramers associate side by side and assemble into filaments. And the inter actions are mostly non covalent, and there is no filament polarity.
Now this structure is tough, flexible and high tensile strength.
Give a specific keratin filament name in the epithelial cells and where they are found
keratin filaments in epithelial cells form a network throughout cytoplasm out of cell periphery.
They are anchored in each cell at cell-cell junctions and connect to neighbouring cells. They provide mechanical strength
What are microtubles involved in?
These are in all organizing functions is all eukaryotes. They are involved in cell organization like vesicle transport, organelle transport and positioning, and animal cells near centromers
They are involved in mitosis and structural support ln cells and motile structures (flagella, cilia)
These are made up of tubulin so they are long and stiff, hence INEXTENSIBLE
How is the structure of the microtubles made?
These are long hallow tubes that are made of subunits of individually two close related globular proteins which are alpha tubulin and beta tubulin. They both first bind to GTP and then bind to each other to form a tubulin hetero dimer.
This regular arrangement gives alpha and beta subunits the polarity, and 13 parallel prototilaments make up a hallow tube
What is the plus end and the minus end in microtubles?
Beta end is the plus end and minus end is the alpha end
What type of bonds are between the individual subunits of microtubles?
Non covalent
Which bonds are weaker in microtubles?
Between protofilaments are weaker than the bonds within each protofilaments
Which side is grow to faster in microtubles
It occurs both sides but is faster on the plus end
When is a microtubule called D-form dimer?
When the protofilaments has been in the tubulin for a while beta tubulin will cut GTP to GDP and that will be called D-form Herero dimer
Is T-form heterodimer growth or shrinkage
T-form is growth while D-form is shrinkage
What is the role of microtubles organizing centres?
In a cell MTOCs are centrosomes in animal cells, the minus end is stabilized at the MTOC and the plus end keeps growing end.
What is dynamic instability?
The plus ends of the microtubles can shrink or grow this is called dynamic instability
When does the GTP cap occur?
If the hydrolysis of GTP is newly added alpha beta tubulin diners is slower than the addition of the a-b- tubulin diners, it leads to the formation of GTP-cap and stabilizes the plus end. And the microtubles continue to grow.
Why is there disassembling in Microtubles?
In GDP tubulin diners; there is conformational change and weaker binding which curves the filaments and disassemble.
What sites does MTOCs have for microtubule growth?
They have Nick eating sites that have gama-ring complexes and accessory proteins. They start assembling new microtubule. The minus end is with gamma-ring complexes and act as attachement sites for the a-beta-tubulin diners
Where do microtubles come from in a non dividing animal cells? In interphase
Most microtubule radiate from one centrosome (MTOS)
