Cell Bio: Final

Cytoskeleton

Cytoskeleton

complex network of filaments that run through the cytoplasm

not static like bone skeleton, is dynamic (constantly changing)

some are tough, some aren’t (intermediate filaments are toughest)

made up of…

1. Actin Filaments - microfilaments - smallest

2. Intermediate Filaments - medium size - includes keratin filaments and interconnect desmosomes

3. Microtubules - largest and tubular - involved in mitosis and radiate from MTOC (microtubules organizing center) in non dividing cells

Functions of Cytoskeleton

gives cell shape

aids movement

serves as a guide for vesicular transport and the orientation of organelles

specific for eukaryotes, not prokaryotes

prime target for treating cancer with many drugs targeting the microtubules that are required for mitotic cells to divide

Actin Filaments

often up to 10% of the total protein of many muscle cells and is most abundant in muscle cells - 1-5% in non muscle cells

molecular weight = 42,000 as Globular-actin

ancient protein given that bacteria, yeasts, and amebas have ancestral actin genes

humans have 6 genes that encode isoforms of actin : alpha, beta, and gamma

Alpha Actin

associated with contractile structures

Beta Actin

at the front or leading edge of moving cells

Gamma Actin

accounts for filaments in stress fibers

F-Actin

consists of units of G-actin arranged in a tight helix with each binding either an ATP or ADP molecule

related to microvilli

one can examine the F to G actin in vitro by changing the concentrations of F actin and ions = addition of magnesium, potassium, or sodium will induce polymerization

in vitro: filaments can treadmill

toxins can alter polymerization

polymerization can be regulated by actin binding proteins

F-Actin Polarity Experiment

myosin 1 has been used to decorate actin and causes the actin to appear as a series of arrowheads pointing in one direction

suggests that f-actin may have an end to end polarity

F-Actin Polarity

has polarity with a positive and negative end that are different from each other and can be monitored by viscometry, sedimentation, or fluorescence

has three phases:

1. nucleation

2. elongation

3. steady state

steady state shows no net change in length because the solution has reached the critical concentration or Cc of G actin

Critical Concentration of G Actin

the concentration of actin at which there is no net change in length of the filament at the positive or negative end

CC+ is - 0.12 uM

CC- is - 0.60 uM

there is a 5 fold difference between the two ends, G actin adds five times faster at the positive end compared to the negative end

Actin Treadmilling

a process that occurs at CC values intermediate between the negative and positive values

in vivo, this process may be responsible for cell movement

occurs in microfilaments and microtubules

Cytochalasin D

fungal product that inhibits polymerization of F- actin by binding to the negative end

Phalloidons

toxins from a mushroom (angel of death) that promote polymerization

one remedy is to eat lots of raw meat that saturates the poison

can be used as a fluorescent tag for labeling actin

stop microfilament activity

Thymosin Beta4

calculations suggest that based on in vitro experiments all the G actin should be in the F actin form - up to 40% is in the G form in cells

a sequestering protein

a small protein that sequesters ATP-G actin and maintains an active G actin pool, the more of this protein in cells the more active the G actin is

Profilin

binds ADP actin in a 1:1 relationship

promotes F-actin assembly by encouraging the ATP for ADP exchange - the only protein known to do that

also aids in the addition of monomers to the positive end of F-actin

Cofilin

binds to filaments containing ADP-actin, inducing them to fragments and thus enhancing depolymerization by making more filament ends

one of many severing proteins

Cofilin and Gelsolin

severing proteins

cap the positive end and therefore prevent adding units, the negative end then shortens, solubilizing the filament

discovered by amoeba where during movement, cytosol moves from the center of the cell to a leading edge, during so it goes from to a sol to a gel

Actin Capping

capZ binds to the positive end and prevents the addition or loss of units from that end

tropomodulin caps at the negative end

if both ends are capped, the filament is stabilized

Formin

nucleates actin filaments

helps in microfilament assembly

Actin and Myosin

experiment on how optical traps (tweezers) are used to measure the force of a single myosin molecule

use infrared light from a laser and measure the step size and force generated from the interaction

Listeria

a bacterial disease (food poisoning) that binds profilin and organizes an actin filament behind it as seen through fluorescent-phalloidin staining

diaphanous gene is associated with inherited deafness and is a member of the formins family

Intermediate Filaments

toughest of all types of filaments

biology’s answer to rope

number of different types exist coded by 70 different genes

do not contribute to cell motility

more static than other two, somewhat dynamic

• injection of a labeled type 1 keratin found its way into keratin filaments in fibroblasts

5 major types: lamins, keratins, neurofilaments (acidic and basic), and desmin

Opsonization

requires antibodies and F-actin

Lamins

type of intermediate filament

most ubiquitous group

found underneath the nuclear envelope

types A, B, and C (small molecules) play an integral role in cell devision

karyoskeleton - nucleus

Keratins

type of intermediate filament

have an acidic and basic type

expressed in epithelial cells

most diverse type

often specific to cell type, many differentiated cells can be subtyped in this way - remember skin

hair perms work by hydrolyzing the disulfide bonds between hair molecules

Neurofilaments

type of intermediate filament

major part of the axon along with microtubules, can also be used to subtype types of neurons

Epidermolysis Bulosa

disease of the intermediate filaments where there is a blistering of skin - regenerative layer rips due to sheer stress

hereditary and was the reason behind ortec international (company to treat disease)

symptoms are similar to effects of mustard gas so it was used to study a potential cure/treatment for soldiers in the persian gulf war

Plakin

an intermediate filament associated protein that attaches intermediate filaments to other structures such as actin filaments and microtubules

Microtubules

largest of the 3 cytoskeletal elements

consists of 13 pro filaments containing alpha and beta tubulin dimmers - 55,000 weight each and found in all eukaryotes, also has several isoforms present

usually singlet, can be doublets or triplets

c. elegans have 11 or 15 protofilaments

has a positive and negative end - treadmilling occurs at cc value

GTP Bound to Alpha Tubulin

non exchangeable

GTP Bound to Beta Tubulin

exchangeable

Microtubule Assembly

similar to F-actin with a nucleation phase followed by an elongation phase

1. doesn’t happen in vivo

2. elongation occurs from a MTOC

3. MAP’s (microtubule associated proteins) catalyzes this assembly in vivo

GTP Gap

disassembly (catastrophe) occurs if the positive end has GDP-beta-tubulin

assembly (rescue) occurs if the positive end has GTP-beta-tubulin

hydrolysis of GTP to GDP can result in work due to energy released

Fish Pigment Cell Experiment

these cells containing vesicles (melanosomes) use microtubules as tracks in melanocytes (pigment containing cells)

cAMP regulates this process but melanosomes don’t migrate it microtubule-destabilizing drugs are used such as colchicine

Experimental Discoveries - Microtubules

• microtubules are often associated with MTOC with centrioles, but not obligatory in this regard

• microtubules are dynamic as evident in cytotoxic T cell after target cell recognition

• microtubules serve as the tracks for neuronal vesicle movement (colchicines treatment)

• flagella regeneration

• assembly/disassembly kinetics

Dynamic Instability Model

describes microtubules growth and shrinkage (catastrophe and rescue)

MAP’s

microtubule associated proteins

part of the microtubule system

co-purify with microtubules

2 types

1. microtubule stabilizing proteins (MAP 1, 2, 4, etc.)

2. microtubule destabilizing proteins

Kinesin 13

family binds to the positive end and makes protofilaments curve, increasing catastrophe’s (disassembly)

don’t have motor activity, hydrolyze ATP at both ends and thus promote depolymerization

OP18/Stathmin

originally discovered as an oncoprotein in cancers, also binds to protofilaments increasing catastrophe’s (disassembly)

Katanin

severs from MTOC’s thus exposing the more labile (unstable) end of the microtubule

TAU Experiment

introduce tau antisense obligonucleotides to neurons - axons don’t form but dendrites do

introduce MAP-2 antisense into neurons - dendrites reabsorb but axons form

Colchicine

an important microtubule drug

has been used along with its relative and colcemid to synchronize cells at the metaphase

has been used to treat gout bc it destabilizes WBC microtubules so that they cant migrate to the site of inflammation

Taxol

an important microtubule drug

extracted from the bark of yew trees

taxotere is a synthetic relative used to treat cancer - approved to treat breast, ovary, and pancreatic cancers

disrupts the M phase, targets microtubules

also called paclitaxel, taxotere is also called docetaxel

Molecular Motors

interact with microtubules to help move vesicles along pathways - “walking down a microtubule”

investigated originally in neurons where both axonal anterograde and retrograde movement has been observed

fastest - 3um vesicles migration is mediated by microtubules

Kinesin 1,2

moves vesicles usually but not always negative to positive

ex: anteroretrograde in neurons

Kinesin 5

can pull on adjacent microtubules

Dynein

moves vesicles positive to negative

ex: retrograde in neurons

needs dynactin to bind to cargo

carries kinesins back on organelles so that kinesin can power the anterograde transport once again

Kartagener’s Syndrome

also called primary ciliary dyskenesia

defect in dynein arms in cilia leading to excess mucus buildup in lungs and bronchial pathways among other problems

Microtubules Diseases

many neurological diseases may be due to defective microtubules because they are required during embryogenesis and development for axonal growth

Uses for Stem Cells

increased understanding of how diseases develop

cure diseases

test new drugs for safety

generate new stem cells to replace or aid diseases or damaged cells

research how certain cells develop into cancer

regenerative medicine applications

fix genetic diseases in the future

tissue engineering

clean meat industry

Stem Cell

a cell that can renew (divide) or differentiate

above controlled stem cell “niche”

number of doublings influenced by source or type : hESC and iPSC’s immortal, adult sourced 50-100-200 doublings (approx)

Adult Stem Cells

most popular are adipose (fat) derived mesenchymal stem cells (adMSCs) now in more than 700 stem cell therapy trials globally

Fetal Stem Cells

amniotic, umbilical cord, placental

Embryonic Stem Cells

hESC’s and hPSCs with hESCs in clinical trials as of 2010

Induced Pluripotent Stem Cells

not in clinical trials in US but patients being treated in japan and australia

Differentiate

cell becomes more specialized such as a fibroblast or hepatocyte

can be partial or full so critical and accepted molecular metrics need to be in place to compare one iPSC generated hepatocyte to another

some stem cells have restricted lineage and are often called progenitor cells because they are limited to only one or two types of cells while others are totipotent

Transdifferentiation (Direct Reprogramming)

ability of a differentiated cell to become another type of differentiated cell without going through an embryonic step

first done experimentally but several cells have been generated since that time

ex: unlike iPSCs

Dedifferentiation and Redifferentiation

ability of a cell to become more embryonic-like and differentiate into another cell type

chemicals like reversine can induce dedifferentiation

demonstrated in the eastern red spotted newt - regenerate lost limbs and eye lenses

Stem Cell Niche

also called stem cell microenvironment

critical to controlling cell division vs differentiation

complex and includes…

1. neighboring cells

2. extracellular matrix

3. local growth factors (FGF)

4. physical environment (pH, oxygen tension, pressure)

Totipotent

all cell types

highest level of “stemness”

Pluripotent

many cell types

restricted stemness

Multipotent

several cell types

stemness even more restricted

Unipotent

one cell type only

Blastocyst

late pre-implantation stage embryo

hESCs originate from inner cell mass

Chimera Test

can determine if a stem cell is totipotent

legal with mice but not humans

we can never prove that any human stem cell derived or isolated in the lab is truly totipotent

only true test for totipotency for a candidate stem cell

label stem cell with a GFP (green fluorescent protein)

implant GFP labeled test stem cell in blastocyst and then implant chimeric embryo in surrogate mother - track the GFP in all tissues and organs of the newborn

Biodistribution and Homing

ability of stem cells to find “home” or its targeted tissue

damaged or compromised tissue releases factors that cause endogenous MSCs to home to damaged sites

occurs in vivo: transplanted XX hearts in XY patients have XY cardiomyocytes upon autopsy (10%) - a clear demonstration of endogenous stem cell homing and repair

Shinya Yamanaka

revolution in stem cell research in induced pluripotent stem cells (iPSCs)

nobel prize winner

STAP

stimulus triggered acquisition of pluripotency - stress turning normal cells into pluripotent undifferentiated cells

made by decreasing pH of medium with acid

reported but now retraced for fraud

Fusogenic

problem with stem cells

can spontaneously fuse with each other forming a tetraploid cell (could generate cancer stem cells)

when injected into patients mechanical stress can cause fusion

Bioethics

the norms of conduct

relative term and country dependent

Therapeutic Cloning

production of embryonic stem cells for the use in replacing or repairing damaged tissues or organs = under laboratory conditions

achieved by transferring a diploid nucleus from a body cell into an egg whose nucleus has been removed

can be used to treat diseases like diabetes and alzheimers

Reproductive Cloning

deliberate production of genetically identical individuals, each newly produced is a clone of the original

develops under uterine conditions

used for embryonic development

SCID (Severe Combined Immunodeficiency) Mice

have no B and T cells and have a compromised immune system

used for determining if an injected candidate stem cell can differentiate in vivo into a multitude of tissue and cell types

also used to determine if a candidate human cancer cell can generate tumors in vivo

Ways to Generate Stem Cells in the Lab

1. somatic cell nuclear transfer (SCNT)

2. parthenogenesis (hPSCs)

3. induced pluripotent stem cells (iPSCs)

Somatic Cell Nuclear Transfer (SCNT)

first done by sir ian wilmut cloning dolly and john gurdon cloning frogs

1000s are required for one implantable embryo - a problem with this process

first pet clone was “little nicky”

as a result of problems, many researchers left the field

some are still attempting human cloning because hESCs could serve as an autograft

important to future developments of iPSCs because it was obvious that cytoplasmic factors could reprogram a somatic nucleus

Dolly the Sheep

born at edinburgh university - first cloned sheep

was euthanized at age 6 due to lung disease and advanced arthritis

other identical clones born from the same SCNT are fine!

monkeys cloned soon after this success

SCNT showed that a somatic nucleus could create an entire functioning animal due to cytoplasmic factors in the nucleus

Experimental Parthogenesis

artificial was first shown by leob using two experimental systems… - reproduction from an ovum without fertilization

1. unfertilized sea urchin eggs were induced to undergo this by changing the osmolarity of the surrounding medium

2. unfertilized starfish eggs could do the same thing with dilute acid

Benefits of hPSCs

only 200 to 300 eggs would be required to generate these cells that could match anyone in the world

Limitations of hPSCs

all alleles will be homozygous because of no sperm

have identical alleles

not FDA approved in the US

is it ethical to create a human embryo?

RT-PCR

true differentiation in culture as revealed by appropriate markers

SCID Mouse Test

the iPSCs from tetramas in mics - a non malignant mass of differentiated cells derived from iPSCs

is a tetramoa generated by iPSC injection?

Enucleate

removing the nucleus from a cell

Anucleate

a cell lacking a nucleus

Function of iPSCs

basic research on differentiation

makes cells more embryonic like

can make patient specific cells of individuals carrying genetic defects

source of cells in the future for stem cell therapy - not yet FDA approved by in clinical trials in the UK

have proven useful in tissue engineering organoids

Pros and Cons of SCNT

could be useful for an autologous (comes from patient) transplant if FDA approved

no US federal laws banning therapeutic or reproductive cloning but some states forbid it

is it ethical? a human embryo is genetically created?

Pros and Cons of Parthogenesis

can match to a world population - only 300 eggs required

all alleles are homozygous, not heterozygous

allogenic (comes from another person), not autologous like SCNT unless female donated egg

is it ethical? a human embryo is genetically created?

Pros and Cons of iPSCs

no human embryo created like previous two

can be autologous or allogenic

but potential for tetracarcinomas

more pluripotent than fat (adipose) - derived adult mesenchymal stem cells and easier to procure

Tumorigenicity

stem cells have long telomeres and can divide many move times than normal cells (telomeres = mitotic clock)

propensity to form tumors and teratomas

one clinical trial started in japan overseen by RIKEN institute was stopped after only one patient due to this concern

Immunogenicity

propensity to trigger immune response

the more frequent the stem cell injections the higher the chance of immune rejection complications that could include anaphylaxis

autologous as well as allogenic can launch as immune response

Inappropriate Differentiation

risk of stem cells differentiating into cells that were not intended and not native to target organ

ex: woman injected with human mesenchymal stem cells (MSCs) near her eyes ended up with bone tissue growing inside her eyelids

Regenerate

the intestinal epithelium can…

Hematopoiesis

blood cell collection and circulation process

includes immune cells, red and white blood cells, and platelets

can also create bone marrow cells, namely erythrocytes, platelets, granulocytes, lymphocytes, and monocytes

Placental Cord Blood

can be used for blood replacement and stem cell therapy

ex: viacord company for public cord blood

Private Blood Cord Bank

incorporated as a “for profit” organization

donors pay an initial fee and a maintenance fee

cells not available to the public

better if there is a genetic disease in the family and multiple members require cells

Public Blood Cord Bank

incorporated as a “not for profit” organization

available to the public through the national marrow donor program through which cord blood is matched

Benefits of C. Elegans

easy to grow on agar plates

non-pathogenic

translucent - can optically section through organism

stable mutants are available to study

all cells have been coded and differentiation predicted

cell division/differentiation patterns can be predicted and always follow the same pattern

many genes like the apoptotic genes have mammalian homologs

apoptotic genes were first identified in this organism by robert horvitz

compromised of a limited number of cells (about 1000)

first mircoRNA (miRNA) discovered = lin-4-RNA

Par Proteins

establish polarity in c. elegans

Types of Cells Without a Nucleus

1. red blood cells = odd shape next to capillary wall which increases CO2/O2 exchange = anucleate

2. epidermis (skin) = increases barrier membrane properties

3. lens epithelium (lens fiber)

Nuclear Pores

protein/mRNA exchange

proteins less than 62,500 daltons can pass

others like… receptors and ATP

Nucleoplasmin

10% of the protein in xenopus laevis eggs

1st molecular chaperone discovered

function: enome stability, nucleosome assembly, and transcriptional regulation

165,000 daltons

ATP regulator dependent