Cell Bio Final

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Cytoskeleton

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is a dynamic system made of protein filaments. There are three classes of cytoskeleton: microtubules, microfilaments (actin filaments), and intermediate filaments. All are filamentous protein polymers made up of many polypeptide subunits. The monomers of the polypeptides interact non-covalently resulting in the dynamic nature of the cytoskeleton.The cytoskeleton is mostly cytoplamic and serves diverse functions such as:
· Cell shape (microfilaments)
· Mechanical support (intermediate filaments)
· Cell-cell adhesion
o Desomosomes (intermediate filaments)
o Adherin junctions (Micro filaments)
· Cell to Ecm attatchmetns
o Focal adhesions (MF)
o Hemidesmosomes (IF)
· Cell division
o Cytokinesis (MF)
· Mitosis (MT)
· Movement of macromolecules and organells
o Chromosomes (MT)
o Vesicles (MT)

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There are three classes of cytoskeleton

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microtubules, microfilaments (actin filaments), and intermediate filaments

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The cytoskeleton is mostly cytoplamic and serves diverse functions such as:

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· Cell shape (microfilaments)
· Mechanical support (intermediate filaments)
· Cell-cell adhesion
o Desomosomes (intermediate filaments)
o Adherin junctions (Micro filaments)
· Cell to Ecm attatchmetns
o Focal adhesions (MF)
o Hemidesmosomes (IF)
· Cell division
o Cytokinesis (MF)
· Mitosis (MT)
· Movement of macromolecules and organells
o Chromosomes (MT)
o Vesicles (MT)

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Filaments are made from

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profilaments à subunits of polypeptide

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Advantages of numberous polypeptide subunits include:

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1. rapid reassembly in response to environmental changes
2. thermal stability (allows for rapid subunit addition and removal at ends)
3. laternal contacts between profilaments provide mechanical strenght

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techniques for studying the cytoskeleton (5 ways)

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1. microscopy (EM)
a. see subunit structure and individual polymers
2. fluorescent light microscopy
a. indirect immunoflourescence
b. make GFP hybrid proteins
3. digital video microscopy
4. drugs or inhibitors
a. inhibit assembly or disassembly of polymers
5. mutations

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Microtubules

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- center near the nucleus and spread through the cell. Microtubules are monomers with a polymer structure. They are associated with MAPS (microtubule associated proteins)

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Main function of mictrotubules

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is axonemal: cell motility, cytoplasmic: organizing and maintaining cell shape, chromosome movement, disposition and movement of organelles.

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Monomers of microtubules:

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· the alpha and beta heterodimer is always together
· in the cytoplasm there is a pool of free dimmer
· both of the subunits bind to GTP but the alpha subunit is always bound to GTP
· The beta subunit is bound to GTP in the cytosol but GDP when in a microtubule BUT, GDP is not necessary or essencial for incorperatin into a microtubule and in experiments when bound with a non hydrolysable analougue of GTP microtubules still formed

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Polymers of microtubules:

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There are 13 profilaments surrounded by a hollow core. There is a plus and minus end (NOT CHARGES). The + end grows and shrinks rapidly and the minus end grows slowely if it is not attatched to anything. These end differ chemically in that the + end had the beta subunit exposed and the – end had the alpha subunit exposed. This results in the differential growth rates.

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For invitro assembly of microtubules you need:

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1. Purified tubulin
2. buffer
3. GTP
4. Mg++

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Kinetic for assembly of microtubules in vitro

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Follows an S curve. In the lag phase dimmers form into oligomers. The elongation phase shows exponential growth. In the plateau for every dimer that is attatched one falls off.

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Microtubule assembly in vivo:

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The MTOC –microtubule organizing center is found in eukaryotic cells. It is found in the basal body for cilia and flagella and the centrosome. The MTOC from the basal body can be isolated and used in in vitro experiments to change assembly kinetics. The kinetic turn into a hyperbolic function, skipping the lag phase.

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Basal body

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has a + end and a – end and tubulin comes out from either side

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Centrosome

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has microfilaments coming in a circular direction all around and does NOT have a plus or minus end. The minus eds of the microtubules are all embedded in the centrosome and all of the growth and shrinkage happends at the plus ends.

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Critical concentration of tubulin dimers

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a number that describes the concentration above which there is no net addition of dimmers. Below this concentration there is net loss of microtubules. The critical concentration is lower at the + end than at the – end. Tredmilling of dimmers occurs at the crit concen. (this is only possible in vitro without a MTOC)

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MTOC

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serves as a site at which MT assembly is initiated and acts as an anchor for one end of these MTs. A centrosome is a type of MTOC. The minus ends of MT are always anchored in the MTOC. There is a limited number of anhchor sites for MT in the MTOC that control how many MT can form

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MAPs microtubule associated proteins

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a variety of proteins that modulate MT assembly, structure, and function. MAPs may bind along the MT and form projections from the wall allowing interaction eith other filaments and cell structures. They also regulate assembly by stabilizing the plus ends of MT.

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Motor MAPs

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include kinesin ad dynein that use ATP to drive the transport of vesicles and organells or to generate sliding forces between MT. Microtubue doublest slide past each other causing bending. There are two main types of motor maps: ones that cause movement toward the + ends called kinesin and ones that cause movement toward the – ends called dynein.

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Kinesin

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: type of cytoplasmis motor map that causes movement of cargo towars the + end. Atp is required

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Dyenin

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type of cytoplasmic motor MAP that causes movement of cargo towards the – end. Requires ATP. The ATP causes a foot of the dyenin to swing forward.

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Nonmotor MAPs

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control MT organization in the cytoplasm. Important in neurite formation.

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Drug inhibitors of microtubules MEMORIZE

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Cholchicine(from a plant, medo saffron and autumn crocus) It binds to free tubulin and prevents it from adding to microtubules (tubulin can fall off but not add on causing net disassembly)
Vinblastine and vincristine- same as cholchicine
Taxol (from pacific yew-tree) binds and stabilizes microtubules causing them to be not dynamic
Tomoxiffin is used to treat cancer (rapidly dividing cells have a lot of MT activity)

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Cholchicine

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(from a plant, medo saffron and autumn crocus) It binds to free tubulin and prevents it from adding to microtubules (tubulin can fall off but not add on causing net disassembly)

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Vinblastine and vincristine

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It binds to free tubulin and prevents it from adding to microtubules (tubulin can fall off but not add on causing net disassembly)

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Taxol

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(from pacific yew-tree) binds and stabilizes microtubules causing them to be not dynamic

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Tomoxiffin

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is used to treat cancer (rapidly dividing cells have a lot of MT activity)Prevents MT activity?

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Axonemal microtubules

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include the highly organized, stabke microtubules found in specific subcellular structures associated with cellular movement, including flagella, cilia, and the basal bodies to which those appendages are attatched.

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Cytoplasmic microtubules

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a more loosely organized dynamic network.They form the mitotic and meiotic spindles and are necessary for chromosome movement, they maintain cell shape and organization and movement of organelles such as vesicles.

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Model for microtubule assembly

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1. at the start of the neucleation process, several tubulin dimmers can aggregate into clusters called oligomers
2. some oligomets go on to form linear chains of tubulin dimmers called porfilaments
3. the porfilaments can then associates with each other side-by-side to form sheets
4. sheets containing 13 or more porfilaments can closeinto a tube, forming a microtubule
5. elongation of th emocrotubule continues by the addition of tubulkin subunits at one ot both ends

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Microfilaments (actin filaments

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line epithelial cells. Some are located just underneath the nuclear membrane, at cell-cell junctions (desmosomes), ECM-cell connections (to linker proteins), at tight junctions. The form the cell cortex. The average diameter of a microfilament is 7nm its monomer is G actin and the gene for this are highly conserved among species

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Main function of microfilaments

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muscle contraction, amoeboid movement, cell locomotion, cytoplasmic streaming, cell division, maintaining cell shape

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Gene conservation experiment for microfilaments (actin filaments)

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Make in vitro transcrips and translations of actin genes from different species. Mix actin from two species (yeast and humans) to make a hybrid filament. Positive control: just human actin Negative control: change pH to prove that the system represents what is happening in vivo. The hybrid filament forms just fine.

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Prokaryotes and actin like filaments

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In 2000, actin like filaments were discovered in prokaryotes which are thought not to have a cytoskeleton. The MREB gene in Bacillus subtilus regulates the shape of the rod bacteria. EXPERIMENT: make KO of MREB gene. Result is round bacteria

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Eukaryotic actin

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is made of G-ctin. It is globular and x ray crystallography shows a U shape with a neucleotide binding site for ATP (when alone) or ADP (when bound). Again this is not necessary for assembly. When G actin is polymerized it is called F-actin. Actin is two strans twisted into a helix. The + end is the barbed end and the minus end is the pointed end. Other molecules involved with actin assembly, disassembly, and stability are usually proteins and lipids

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Drugs for actin microfilaments

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Cytochalasins are fungal metabolires the inhibit GàF polymerization
Latruncutins came from the red sea sponge and also inhibit polymerization of actin
Phalloidin form the death cap mushroom stabilize filaments and do not allow disassembly. It binds to intact microfilaments and stabilizes them. A fluorescent tag can be added to the molecule for study. The cell dies.

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Cytochalasins

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are fungal metabolires the inhibit G to F actin polymerization

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Latruncutins

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came from the red sea sponge and also inhibit polymerization of actin

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Phalloidin

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form the death cap mushroom stabilize filaments and do not allow disassembly. It binds to intact microfilaments and stabilizes them. A fluorescent tag can be added to the molecule for study. The cell dies.

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Microfilaments in epithelial cells

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Can be found in microvilli. The bundles of MF maintain structure. The – ends are anchored at the base of the microvilli in the tetrminal web. The + ends are at the tip.

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Actin binding proteins

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bundle actin together

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Lateral crossinkers

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attatch actin to microvilli

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Microfilaments in erythrocyetes (RBC)

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are abundant in the cortex region of the cytoplasm just below the plasma membrane.

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Intermediate filaments

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there are many different types of intermediate filaments coded for by many diff genes (especially in vertebrates) for diff tissues at diff developmental stages. The central domain of IF is highly concerved and codes for an alpha helix. The carboxy andamino terminus lengths and sequences determine the differences in filaments. They are usually 8-12nm in diameter, have no known polarity.

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The main functions of intermediate filaments

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is structural support, maintaining cell shape, formation of the necluear lamina a scaffolding, stenghthening of nerve cell axons, and keeping mucle fibers in register.

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Assembly of intermediate filaments

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two monomers assemble into a dimer amino ends together. Then dimmers assemle linearly C to N end and then in a
X
X
X
Pattern.

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Intermediate filaments and cancer

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in the 1980’s it was found that Intermediate types are tissue specific and can be used for tumor typing:
A biopsy of the tumor is perfumes
IF are ID with antibodies
The ID can show what tissue the tumor came form
Since treatment depends highly on tissue type, this improves the diagnosis.

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Nuclear lamins

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IF in nuclear membrane are there to maintain nuclear shape.

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Motility

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Motility occurs typically with the help of MT based cilia and flagella, relatively permanent structures that are typical in eukaryotic cells.

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Cilia

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bundles of MT surrounded by plasma membrane 2-10 microns long

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Flagella

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bundles of MT surrounded by plasma membrane 10-200 microns long. A cross section of the flagell shows a 9 + 2 structure (two bundles on the inside and 9 on the outside.

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Function of cilia

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in unicellular protozoa with cilia the cilia collect food. In multicellular organisms some cell (like lung cells) have cilia to move material past cells.

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Role of kinesins in vitro experiment:

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Need MT, ATP kinesin and polystyrene beads to act as cargo. Optical tweezers were used to pick up beads and but them on the microtubule. Digital video microscopy showed movement of the beads. If a basal body is added, anchoring the – ends, beads are seen ,igrating away from the basal body towards the + end.

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Movement of cargo in cells

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Dyenines move cargo from the RER past to the Golgi towards the MTOC towars the – end of MT.
Kinesin move cargo from the Golgi to the membrane, away from the MTOC, towards the + end of the MT

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Actin based movement in non muscle cells

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There are 4 types of movement based on actin:
1. cytoplasmic streaming used by plants and algae
2. movement of pathogens thorugh host cells
3. cell crawling
4. ameboid movement

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actin structure and movemen

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actin filaments use the myosin family of proteins as motor proteins

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Myosin

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a super family of proteins that bind ATP and cause movement. There are multiple polypeptides that bind together and can displace mictofilamets in respect to one another.

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Cytoplasmic streaming

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movement of cytoplasm inside the cell around the vacule based on actin and myosin.

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Cytoplasmic streaming experiment

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1. take latex beads and coat htem with myosin
2. break open algae cells (using a French press)
3. view using digital video microscopy
a. deplete ATP à no movement
b. add cytochalasin (which disrupts actin filaments à no movement

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movement of infectios organisms through cell

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example: lysteria a human pathogen that causes food poisoning. It can be found in uncooked cold cuts, hummus, and unpasturized cheese. It uses comet tails of actin to propel itself through cells and infect other cells

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cell crawling

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requires ATP and delivery of G actin because diffusin is not fast eough

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filipodia

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very common) used bundles of microfilaments. Small finger like projections of cytoplasm from a cell. Parallel arrangement of actin

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lamellopodia

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sheet like extensions of cytoplasm from a cell. Has a network arrangement of actin.

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Regulating the structure and arrangement of actin in the cell :

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depends on small G-proteins such as RAC, Rho and CDC42.
Rac- g protein that regulated lamelopdia
Rho g protein that regulates stress fibers
Cdc42 g protein that regulates fillipodia

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Rac

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g protein that regulated lamelopdia

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Rho

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g protein that regulates stress fibers

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Cdc42

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g protein that regulates fillipodia

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Nucleus

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two phospholipid bilayers, nuclear pores for import and export of materials, nuclear envelope, nuclear lamina, nuclear matrix, surface is studded with ribosomes

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There are two types of chromatin

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1. heterochromatin is associated with non active genes
2. euchromatin is associated with actively transcribed genes

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nuclear pores

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allow import ad export of materials. Can cound number using freeze fracture. There are usually 3000-4000 in a mammillian cell in a nucleus. Ions move by diffusion in and out of the pores but larger molecules move by selective active transport. Molecules that are larger have a neuclear localization signal amino acid sequences.

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NLS

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nuclear localization signal) is necessary and sufficient for import into the nucleus. If yo ucaot larger gold particles with the NLS they get through

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NLS import steps

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1. a protein in the cytoplasm has an NLS signal
2. an importin with a domain that bind with NLS forms a complex with the protein
3. import is allowed in
4. once insode GTP Ran removes the importin
5. GTP ran and the importin leave the nucleus
6. importin is released and is ready to bind to a different nuclear protein with an NLS

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experiment to determine size max of diffusion through pores

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make colloid gold of dif sizes and inject into cell. Saee what gets into the nucleus. Up to 10 nm max for diffusion. Or use radioactively labled proteins. Only 20,000 D (small proteins get in)

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nuclear lamina

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is just inside the nuclear envelope. It contains a cytoskeleton based structure based on intermediate filaments. It is important for the structure of the nucleus. It is assembles in interphase and disassembled in mitosis.

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Nuclear matrix

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where chromain is outside of the nucleolus

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What goes in and out of the nuceus

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(through the nuclear poses)
In: transcription proteins and polypeptides important in the nucleus such as dan and rna polymerases. Histones also need to go in. In s phase there is a need for 300,000 histones meaning that there are about 100 histones moving through each pore every minute.
Out rRNA, tRNA, mRNA

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Nucleolus

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is electron dense, is the site for ribosome subunit assembly. It has no membrane, it is continuous with the rest of the nucleus.

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Experiment to determine the activity of the nucleolus

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Radioactively lable RNA with radioactive uracil. Do tem and audoradiography, see spots on nucleolus. This is the suggested site of ribosome assembly.

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Nuclear lamins

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– a type of intermediate filament that constructs the nuclear lamina. They provide structural support for the nuclear membrane and may be sites for chromatin attatchement.

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Nuclear lamins and disease

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Progeria or rapid aging in children may be cause by defective nuclear lamins due to a single base pair mutation. In the nuclei of patients 50% are misshapen

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Cell cycle The

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The cell cycle is separated into two main parts, M phase which includes mitosis and cytokinesis, and interphase, which consist of G1, S, and G2 phase. The length of a cell cylcle varies. A typical mammalian cell cycle is 18-24 hours. Some cells such as ecoli, blood stem cells, and early embryonic cells have a very short cell cycle. Other cells only divide when stimulated to. Examples of these kind of cells include liver cells that can regenerate a part of a liver after trandplant donation and lymphocytes that divide when the mmune system is stimulated. Some cells such as nerve cells or red blood cells don’t divide at all.

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How to determine the length of the cell cycle?

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Count cells and see how long it takes for the cells to double in number.

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M phase

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part of the cell cycle that consists of mitosis and cytokinesis. Usually 30-45 minutes. The length of M phase can be determined using light microscopy to count the cells. (view mitotic plates, concentrated chromosomes etc…)

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Intermphase

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part of the cell cycle that consists of G1, S and G2 phase.

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G1

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a part of interphase in which cell growth and metabolism occurs. When cells are non dividing G1 is usually called G0. For dividing cells G1 usually lasts 8-10 hrs in a 18-24 hr cycle.

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S phase

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a part of interphase where DNA replication and doubling of chromosomes occurs.

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How to determine the length of S phase

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1. add a lable such as tridiated thiamidine that will be encorperated into new DNA
2. pulse
3. determine what % of cells are labled
4. in order for the experiment to work the cells must be asynchronous
5. if you assume the cell cycle is 24 hours, and 33% of cell have the lable, than the length of S phase is 24 x .33 = 8 hours

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G2 phase

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a part of interphase where cell growth and preparation for M phase occurs. Usually 4-6 hours

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Experiments about the discovery of M phase and how it works

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There are two general experimental strategies, 1. cell manipulation experiments and 2. genetic experiments that screen for mutants.

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1. cell manipulation experiments to study M phase
a. Cell FUSIONS

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i. induce two cells to fuse together
1. use PEG, electrporation, or viruses such as semlici forest virus or vesticular stomititus virus to induce fusing.
2. A fused cell is called a heterokaryon
ii. fuse two cells at different stages in the cell cycle
1. S cell + G1 cell
a. in heterokaryon, nucleus from G1 is induced to enter S phase
2. G1 cell + M phase cell
a. G1 cell enters directly into M phase

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1. cell manipulation experiments to stufy M phase
b. Cell material injections

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i. Use frog eggs as a model. Take the oocytes which are suspended in G2 phase of meiosis 1 and eggs which are in M phase of meisis 2
ii. Take cytoplasm from one and inject into cell
1. M phase cytoplasm into G2 cell
a. Cell enters M phase
2. cytoplasm from G2 into oocyte (control)
a. nothing happens
3. other controls
a. prick cell with needle
b. inject saline

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2. Genetic Approaches to studying M phase

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a. Lee Hartwell and Paul Norse used yeast as a model.
b. To select for mutants in the cell cycle
i. Induce mutogenesis
ii. Select for conditional mutants (mutants that express the mutant phenotype only under certain conditions)
iii. A common type of mutant found was a temperature sensitive ( either hot or cold mutant)
iv. They screened for cells that grow and divide at one temp (31 degrees for yeast)
v. Shift the temp and look for cells that grow and divide abnormally (at a restrictive temperature)
c. they found a large collection of cell cycle mutant
d. they also found regulatory molecules that mediate cell cycle checkpoints

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1. G1 check point

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a. Is the cell ready for S phase?
i. Right cell size
ii. Nutrients
iii. Growth factors
iv. DNA damage

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b. regulatory molecules in the G1 checkpoint

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i. A protein complex of cyclin and cyclin dependant kinase are responsible for the checkpoint
1. cyclin is only present in large quantit
ies at certain parts of the cell cycle. It must be at a high concentration for the cell to move on to S phase. If everything is fine, the peak of concentration of cyclin is at the end of G1
2. cyclin dependant kinase CDK is always present in the cell
ii. cyclin/CDK phosphorylate a target that results in S phase
1. the target protein in RB/E2F complex. In this form, E2F, a transcription factor is inactive.
2. when cycylin/CDK phosphorylate the complx, E2F detaches and can transcribe genes that lead to S phase.

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ii. cyclin/CDK for G1 checkpoint

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phosphorylate a target that results in S phase
1. the target protein in RB/E2F complex. In this form, E2F, a transcription factor is inactive.
2. when cycylin/CDK phosphorylate the complx, E2F detaches and can transcribe genes that lead to S phase.

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2. G2 check point

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a. Is the cell ready for M phase
i. Cell size
ii. DNA damage

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b. regulatory proteins for G2 checkpoint

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i. G2 cyclin and cyclin dependant kinase are responsible.
ii. They were discovered using the cytoplasmic injection experiments by using a mature egg as a donor for cytoplasm and a recipient oocyte arrested in G1
iii. The oocyte matures into an egg with the help of maturation promoting factor (MPF) which turned out to be a cyclin/CDK
1. MPF is highly conserved
2. human MPF into yeast cells that are defective for MPF causes the yeast cells to mature
3. concentration of G2 cyclin peaks at the end of G2
iv. role of mitotic G2 cyclin/CDK in promotin mitosis
1. phosphorylates histones and chromosomes, which triggers chromosome condensation
2. phorsphoryaltes MAPs which causes spindle assembly
3. phosphorylates lamins, causing nuclear membrane breakdown

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iv. role of mitotic G2 cyclin/CDK in promotin mitosis

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1. phosphorylates histones and chromosomes, which triggers chromosome condensation
2. phorsphoryaltes MAPs which causes spindle assembly
3. phosphorylates lamins, causing nuclear membrane breakdown

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3. M phase checkpoint

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a. Are all of the chromosomes attatched to the spindle?
b. Mediated by mitotic CDK/ cyclin (same as above) which prosphorylates anaphase-promoting complex, a multi protein complex that controls the final phases of mitosis by protmoting the destruction of selected proteins. Anaphase promoting complex is a type of protein called a ubiquitine ligase, which degrades proteins by joining them to a small protein called ubiquitin.
i. Sister chromatids that are not yet attatched to spindle send a signal that they are not ready for anaphase. The signal comes from the kinetichore
ii. This signal inhibits cdc 20 a protein that is required to turn on anaphase promoting complex
iii. After the chromosomes are attatched to the spindle cdc20 helps turn of anaphase promoting complex by binding to it
iv. Anaphase promoting complex is phosphorylated by mitotic CDK- cyclin (MPF) and turned on
v. It degrades the securin that is bound to saparase
vi. Separase degrades the cohesin that binds sister chromatids together
vii. Anaphase begins
viii. Anaphase promting complex also destroys the supply of MPF (by using it up), effectively ending mitosis.

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If a cell does not pass a checkpoint

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the cell cycle is stopped and the cell repairs any damage. If damage cannot be repaired, programmed cell death or apoptosis occurs.

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Role of p53 “tumor suppression gene”

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Damaged DNA turns on a protein called ATM that phosphorylated p53
P53 prevents damaged cells from dividing by causing either apoptosis and/or cell cycle arrest
If p53 is defective/ineffective cancer may occur

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oterrorism threat of an organismis judged on several factors

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1. What is the degree of harm?
a. Infectivity
i. Can it infect humans
1. some animal pathogens can mutate to infect humans (SARS, avian flu)
ii. Is it pathogenic?
1. Does it cause harm?
iii. what is the virulence?
1. measured by CD 50, the amount of pathogen that causes ½ of infected organisms to die
2. a low LD indicates high virulence
iv. what is the transmissibility?
1. mechanisms can be
a. person to person
b. aerosols (flu)
c. contaminated food supply (cholera)
d. vectors (malaria)
v. what is the incubation period
1. the amount of time it takes to show symptoms
2. a long incubation period indicates that high chance the disease will spread rapidly

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Anthrax

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is a gram positive bacterium that can form spores. Anthrax spores are an inert form of the bacteris that can be carried long distances and survive for years. When the proper consitions arise the spores can germinate and infect people and animals.

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There are three types of Anthrax infections with different fatality rates

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1. cutanaceous (via skin) accounts for 95% of cases
a. fatality rate is 20% without treatment
2. gastrointestinal (from animal meat)
a. 50% fatality rate
3. inhalation
a. spores germinare inside macropages, duplicate, travel through lymph and burst cells open

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Cell bio of Anthrax

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The geminated vegetative cell causes the disease
It makes a cap of polyglutamic acid that allows the bacteria to evade the immune system
It makes 3 toxins that are coaded for on plasmids:
Protective antigen (PA)
Lethal factor
A protease that cleaves MAPKK a communication factor that is important in cell cycle progression. It leads to cell death.
Edema factor
Acts as a adenyl cyclase. Makes cAMP and messes up signal transduction in the cell, messing up ion balance and leading to cell swelling.

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Protective antigen (PA)

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) binds to hosts cell surface ad acts as an anthrax toxin receptor.A protease cleaves part of PA. Lethal factor and edema factor then bind to the recptor.

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Lethal factor

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A protease that cleaves MAPKK a communication factor that is important in cell cycle progression. It leads to cell death.

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Edema factor

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Acts as a adenyl cyclase. Makes cAMP and messes up signal transduction in the cell, messing up ion balance and leading to cell swelling.

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How anthrax gets into the cell

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Protective antigen (PA) binds to hosts cell surface ad acts as an anthrax toxin receptor.A protease cleaves part of PA. Lethal factor and edema factor then bind to the recptor. The whole complex is endycytosed. In the acidic envirometn of endosomes LF and EF disassociate and are transported into the cytoplasm.

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Treatment of anthrax

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antibiotic cipro taken for 60 days. The reason for long treatment is that it takes that long for all the spores to geminate so all the spores must be destroyed.