module 5 Flashcards
cytoskeleton is composed of
- microtubules
- actin filaments (microfilaments)
- intermediate filaments
microtubules
In epithelial and neuron cells:
- Support and organelle transport
In dividing cell:
- Form mitotic spindle required for chromosome segregation
Structural characteristics:
- Long
- Hollow
- Unbranched
- Composed of tubulin
found in nearly all euk cell
assembled from tubulin
polarity: (-) end in the middle of the cell, (+) towards the edges of the cell
actin filaments
- Aka microfilaments
- Support of microvilli of epithelial cell
- Part of motile machinery in neuronal elongation and cell division
- Structural characteristics:
- Solid
- Thin structures
- Organized into branched networks
Intermediate filaments:
- Structural support
- Structural characteristics:
- Tough
- Ropelike
- Composed of variety of related proteins
- no polarity
- include neurofilaments, major component of structural framework supporting neurons
Protofilaments:
- Globular proteind arranged in longitudinal rows
- Aligned parallel to the long axis of the tubule
- Held by noncovalent interactions
- Assembled from dimeric blocks of one alpha and 1 beta tubulin
- Assymetrical: alpha on one side and beta on the other
- All protofilaments have same polarity:
- Important in growth of microtubules and their ability to participate in directed mechanical activities
- end is beta
- End(-) is alpha
microtubule associated proteins function
- To increase the stability of microtubules
- To alter microtubules rigidity
- Influence the rate of microtubule assembly
- Attach microtubules to one another maintaining parallel alignment
functions of microtubules
- Support of the cell
- Ciliary and flagellar motion
- Movement of material b/ cell body and axon terminals
- Mitosis/meiosis
- Intracellular organization etc.
- Structural support and organizer:
- Distribution of microtubules helps to determine the shape of the cell
- Influence on formation of cell wall
- Maintaining the internal organization of the cells
- Intracellular mobility:
- Transport of material from one membrane compartment to another
- Movement of vesicles along the axon in both directions
- Serve as tracks for variety of motor proteins
- Associated w/ cytoskeleton convert chemical E into mech E that is used for cellular movement
- Move unidirectionally along their track in stepwise manner
- Microtubules moves in the direction opposite to the direction that the motor is stepping
Importance of beta tubulin binding of GTP and subsequent hydrolysis
- Beta tubulin is a GTPase
- GDP after the hydrolysis remains bound to the assembled polymer
- During disassembly: GDP->GTP
- Presence of cap of tubulin-GTP dimer at + end favors addition of more subunits and the growth of microtubule
motor proteins associated w/ microtubules
- kinesin
- cytoplasmic dynein
kinesin
- motor protein associted w/ microtubules
- Structure:
- Tetramer constructed from two identical heavy and two identical light chains
- Pair of globular heads
- Bind microtubule
- ATP-hydrolysing engine
- Neck
- Stalk
- Where H and L chains form DH
- Binds cargo to be hauled
- Tail
- Moves towards + side of the tubule
- Moves along a single protofilament of microtubule proportional to [ATP]
- Moves via “hand-over-hand” mechanism
- Movement is processive:
- One protiens moves along individual microtubule for long distance
- Independent, long-distance transport of small cargo
- Important function during cell division
- Force-generating agents for movement of the transport vesicles (tend to move in outward direction
cytoplasmic dynein
-
Structure:
- Two identical heavy chains:
- Large globular head:
- Force generating engine
- Elongated stalk:
- Microtubule binding site
- Tail:
- Binds intermediate and light chains
- Large globular head:
- Variety of intermediate and light chains
- Two identical heavy chains:
- Moves towards (-) end
-
Functions:
- Positioning the spindle and moving chromosomes during mitosis
- Positioning centrosome and Golgi and moving organelles, vesicles and particles
- In nerve cells:
- Retrograde movement of organelles
- Anterograde movement of microtubules
- Retrograge movement towards the center of the cell
- Responsible for movement of cilia and flagella
- Requires intervening adaptor - dynactin
- Increases processivity of dynein
dynactin
- intervening adaptor for dynein
- increases processivity of dynein
Functions of MTOCs:
- They are variety of specialized structures that initate microtubule nucleation and organize microtubules
- Control # of microtubules, their polarity, # of protofilaments that make up their walls and the time and location of their assembly
What are 2 different MTOC:
Centrosome:
- In animal cells
- Place from which newly assembled microtubules grow in all directions
Basal bodies:
- Where microtubules of cilium and flagellum originate
How cilia and flagella can undergo their bending movements:
-
Dynein arms act as swinging cross-bridges that generate the force required for movement
- Steps:
- Dynein is tighly anchored to outer surface of tubule A and its globular heads point towards B tubule
- Dynein arms anchored along tubule A of lower doublet attach to binding sites on tubule B of upper doublet
- Power stroke: lower doublet slides towards basal end of upper doublet
- Dynein arms detached from tubule B
- Arms have reattached to the upper doublet so that another cycle can begin
- Steps:
- Sliding-microtubule mechanism of ciliary or flagellar motility:
- When cilium is straight: all outer doublets end at the same level
- Cilium bending occurs when doublets on the inner side of the bend slide beyond those on the outer
Sliding-microtubule mechanism of ciliary or flagellar motility:
- When cilium is straight: all outer doublets end at the same level
- Cilium bending occurs when doublets on the inner side of the bend slide beyond those on the outer
distinct characteristics of intermediate filaments
- Chemically heterogeneous
- Assembled in tetramers formed by two antiparallel dimers
- Lack polarity
- Resist tensile forces
Role in IF in neurons:
- IF include neurofilaments - major component of the structural framework supporting neurons
- As the axon increases t=in diameter, the neurofilaments provide support
8 actin binding proteins and their functions
Nucleating proteins:
- Form template to which actin monomers can be added
Monomer-sequestering proteins:
- Bind to acti monomers and prevent them from polymerizing
End-blocking proteins:
- Reglate the length of actin
Monomer-polymerizing proteins:
- Stimulate actin polymerization during cell locomotion
Actin filament depolymerizing proteins :
- Enhance depolymerization
Cross-linking proteins:
- Promote formation of loose networks of filaments
Fimalent severing proteins:
- Decrease the viscosity of the cytoplasm
Membrane-binding proteins:
- Bind to the plasma membrane and allow it to protrude or invaginate from the cell
Nucleating proteins:
- Actin binding proteins
- form template to which actin monomer can be added
monomer sequeatering proteins
Actin binding proteins
binding actin monomers and prevents them from polymerizing
end-blockin proteins
Actin binding proteins
regulate length of actin
monomer polymerizing proteins
Actin binding proteins
stimulate actin polymerization during cell locomotion
actin filament depolymerizing proteins
Actin binding proteins
enhance depolymerization
cross-linking proteins
Actin binding proteins
promote formation of loose networks of filaments
filament serving proteins
Actin binding proteins
decrease viscosit of cytoplasm
Cellular activities involving motility of nonmuscle cells in which actin filaments are involved:
Cytokinesis
Phagocytosis
Cytoplasmic streaming
Vesicle trafficking
Cell-substratum interactions
Cell locomotion
Axonal growth
Chnges in cell shape
Actin filament assembly/disassembly:
- ATP is important
- Barded end incorporates monomer at faster rate
- Barded and pointed ends require different [ATP-actin monomer] to elongate:
- Barded needs less
- Steps:
- Add preformed actin filaments to the solution of actin in the presence of ATP
- If [ATP] is high, actin is added at both ends
- Addition continues only at the barded end because [ATP] is only high enough for addition at that end
- Addition continues at the barded end, but loss of the monomer occurs at the point end to respond to the decrease in free monomers
- Addition/removal rates change to maintain free monomer concentration stable
general structure of myosin
- Head:
- Contains:
- Site that binds an actin filaments
- Site that hydrolyses ATP to drive the mysosin motor
- Contains:
- Tail:
- Divergent and different in various types
- Variety of light chains
Conventional (type II) myosin
- Primarily motors for muscle contraction
- Can be found in non-muscle cells:
- Move towards barded end
- Function in non-muscle:
- Split the cell into two
- Generate tension at focal adhesion
- Cell migration
- Ends of tails point towards the center of the filament and the globular heads point away from the center
Unconventional myosin:
- Subdivided into 17 different classes
- Contains single head
- Unable to assemble into filaments in vitro
- Can exert tension on plasma membrane
- Unable to form filament
- Operate as inidivual proteins
Stages of the cell cycle:
- Interphase:
- G1:
- Cell grows and carries out normal metabolism
- Organelles duplication
- S phase:
- DNA replication
- Chromosome duplication
- G2:
- Cell growth
- Preparation for mitosis
- G1:
- Mitosis:
- PMAT:
- Duplicated chromosomes are separated into 2 nuclei
- cytokinesis:
- Division of the entire cell into 2 daughter
- cytokinesis:
- PMAT:
Cell cycles vary among different types of cell depending on the cell type and environmental conditions
important regulation stage (check points) of cell cycle
@ end of G1:
- First transition pt START
- If pass-> commited to the DNA replication
- Uses Cdk
@ end of G2:
- Uses mitotic cyclins
Middle of mitosis:
- Determines if they’ll complete cell division of reenter G1 of the next cycle
checkpoint control
- Ensures that DNA is not damaged
- Stop the progress of cell cycle if:
- DNA is damaged
- Critical processes have not been properly completed
- Activated by the system of sensors that recognize DNA damage or cellular abnormalities
- Cell stops its progress at one of those check points via Cdk inhibitor
G0 stage:
- Cells that are arrested in the state of no cell division (temporarily or permanent) preceding the initiation of DNA synthesis
- Ex. nerve cells, muscle cells, RBC (b/c highly specialized and lost the ability to divide)
How is the activity of cyclin-dependent kinases regulated:
- Cyclin dependent kinases are enzymes that phosphorylate other protein and regulate cell cycle
- Regulated by:
- Presence of specific cyclins:
- Required subunits for specific Cdk activities
- Levels of cyclins that vary throughout the cell cycle partially controlling the activity of Cdks and secondarilly controlling the activities of the substrates phosphorylated by those specific Cdks
- Other kinases, which phosphorylate them result in activation/deactivation depending on state of phosphorylation
- Phosphatases dephosphorylate them
- May inhibit/activate some Cdks
- Subcellular localization may separate Cdks from their substrates or create microenvi for them
- CDK inhibitors
- Cdks can be targeted for degradation (ex. by ubiquitin-proteasome pathway)
- Presence of specific cyclins:
Prophase
- Chromosomes condense
- Formation of compact mitotic chromosomes
- Chromosomes composed of two chromatids attached together at centromere
- Golgi and ER fragment
- Nuclear envelope dissapeares
prometaphase
Chromosomal microtubules attach to the kinetochores pf chromosomes
Chromosomes are moved to spindle equator
Metaphase:
Chromosomes alligned at metaphase plate
Chromosomes attached to both poles by the microtubules
anaphase
Centromere splits
Chromatids separate
Chromosomes move to the opposite spindle poles
Spindles poles move further apart
Telophase:
Chromosomes cluster at opposite spindle poles
Chromosomes become dispersed
Nuclearenvelope assembles around chromosome cluster
Golgi and ER reform
Daughter cells form by cytokinesis
centromeres
- Primary constrictions in chromosomes
- Residence of highly repeated DNA sequence
- Serves as binding site for specific proteins
- Has kinetochore:
kinetochore
- On the outer surface of centromere of each chromatid
- Proteinaceous
- Button-like
- Function:
- Residence of motor proteins involved in the chromosome motility:
- Dynein
- CENP-E
- Depolymerase
- Site of attachment of chromosome to the dynamic microtubules of mitotic spindle
- Key component in the signalling pathway of an important mitotic checkpoint
- Residence of motor proteins involved in the chromosome motility:
centrosome
initiate microtubule assembly that is responsible for formation of mitotic spindle
Tubulin flux
- Net addition of tubulin at + end (@ kinetochore side)
- Net loss of tubulin @ - end
- Subunits move along the chromosomal microtubules from kinetochore towards the pole
- happens during metaphase
anaphase promoting complex (APC)
- Adds ubiquitin to proteins at different stages of the cell cycle targeting them for proteolysis
- Adaptor protein:
- Determines which proteins serve as APC substrates
- APC-Cdc20:
- APC-Cdh1:1
- The importance of targeted proteasome-mediated proteolysis in progression thru mitosis:
- Completion of mitosis requires the cessation of activity of Cdk1
Cytokinesis:
- In animal cells forms a furrow that moves inwards towards the center of the cell
- In plants: form cell plate:
- Secretory vesicles from Golgi align along the equatorial plane and begin to fuse with one another
- Membrane of the vesicle become plasma membrnae
- Content of the vesicle forms the cell plate separting cells
- Role of actin filaments:
- Becomes assembled in a ring at the cell equator
- Contraction of the ring requires action of myosin -> formation of furrow that splits the cell in two
- Are concentrated in a circular equatorial band within cleavage furrows
purpose of meiosis
Reduce # chromosomes
Increase genetic variability
Prophase I
formation of tetrads (synapsis b/ homologous chromosomes)
cross-over @ chiasmata
movements of tetrads towards the metaphase plate begins
metaphase I
- Two homologous chromosomes of tetra are connected to the spindle fibers from opposite pole
- Sister chromatids are connected to the microtubules from the same spindle
- Side-by-side arrangement of kinetochores
anaphase I and telophase I
Anaphase I:
- Homologous chromosomes separate
- Dissolution of chiasmata by proteolytic cleavage of cohesin
Telophase I:
- Chromosome dispersion, but don’t reach very extended states
- Nuclear envelope may/not reform fully
metaphase II
Kinetochores of sisters chromatids face opposite pole and are atached to the opposite sets of chromosomal spindle
anaphase II
separation of sister chromatids
how do nuclei formed in mitosis and meiosis differ from one another
In mitosis:
- Pait of homologous chromosomes
- Genetically identical to the mother cell
In meiosis:
- Contain one chromosomes from each pair of homologous chromosomes contained by parental cell
- Have the potential to be genetically different from parent chromosomes
Signal transduction:
Process in which info carried by extracellular messengers is translated into changes that occur inside the cell
G-protein:
- GTP-binding proteins
- Associated with:
- Vesicle budding
- Microtubule dynamics
- Protein synthesis
- Nucleoplasmic transport
- Recognized by G protein-coupled receptos (GPCRs) that are composd of transmembrane alpha helices:
- Capable of binding a large variety of ligands
- Active when bound to GTP
Inactive when bound to GDP
Mechanism of receptor-mediated activation of effectors by G proteins
- Ligand binds to the receptor
- Alters receptor conformation -> increases affinity for G protein
- G protein alpha: GDP out, GTP in
- G protein alpha dissociates from beta and gamma
- Alpha-GTP binds to the effector (adenyl cyclase)
- Activation of adenyl cyclase
- Beta and gamma G protein bind to adenyl cyclase
- Activated adenyl cyclase: ATP->cAMP
- GTPase hydrolyses GTP->GDP and deactivates alpha G protein
- G alpha reassociates w/ G beta and gamma
- Adenyl cyclase stops its activity
- GRK ( G protein-coupled receptor kinase) phosphorylates receptor with use of ATP
- Arrestin binds to the receptor inhibiting it from activating more G proteins
- Receptor-arrestin taken up by endocytosis
Protein tyr kinase:
Involved in:
- Regulation of cell growth
- Cell division
- Cell differentiation
- Cell survival
- Attachment to extracellular matrix
- Migration of the cells
Expression of mutant protein tyrosin kinase can’t be regulated and leads to CA
Necrosis:
Less orderly than apoptosis
Swelling of both cell and its internal membranous organelles
Membrane breaks
Leakage of cell content
Inflammation
apoptosis
Neat abd orderly process:
- Overall shrinkage in volume of cell and nucleus
- Loss of adhesion to neighboring cell
- Formation of blebs
- Dissection of chromatin into small fragments
- Rapid phagocytosis
Cell signaling:
- Process by which info is relayed across the plasma membrane to cell interior
- Often this info is transmitted to the nucleus
- Include:
- Recognition of the stimulus
- Transfer of signal across the plasma membrnae
- Transmission of signal to specific effector
- Cessation of the response
Second messanger
- Substance that is released into the cell as the result of binding of the 1st messanger (hormone or ligand) to a receptor outside the cell
- Include:
- cAMP
- IP3
- Ca2+
- DAG
- Nitric oxide
Ras:
- Is a G protein that is part of many pathway controlling cell growth
- Needs to be quickly turned off after activation
- In oncogenes, changes to Ras causes ot to be permanently on ->uncontrolled growth w/t differentiation ->tumor
- Pathways are activated when growth factor bind to the extracellular domain of its receptor
How do the properties of CA cells manifest themselves in culture:
Grow in multilayered clumps
Continue to grow regardless the presence/absence of exogenous growth factors
Unable to respond to inhibitory signals from neighboring cells
What agents are carcinogenic:
- Chemical mutagens
- UV radiatino (forms thymidine dimers)
- DNA- and RNA-tumor viruses:
- Effects are more complicated
- Some can induce transformation b/c they have appropriated cellular genes that can hinder cells’ growth control function
- Inherited mutation
- Diet:
- Can increase or decrease risk of CA
How does CA causing mutation arise?
From DNA damage caused by normal metabolic rnx that the cell unable to repair
Basic properties of CA cells:
- Malignant tumors tend to metastasize
- CA cells have lost their growth control, and have following growth properties:
- Grow in multilayered clumps
- Continue to grow regardless the presence/absence of exogenous growth factors
- Unable to respond to inhibitory signals from neighboring cells
- CA cell often have highly aberrant chromosomes (aneuploidy), while healthy cells have a pair of identical chromosomes
*
genes involved in carcinogenesis
tumor suppressor genes
oncogenes
proto-oncogenes
Tumor suppressor genes:
- Act as cell’s brakes
- Encode proteins that restrain cell growth and prevent cells from becoming malignant
- If copies of the mutatte tumor suppressor genes on both homologous chromosomes -> loss of growth control
- Loss-of-function mutation on both homologous->CA
oncogenes
- Encode proteins that promote the loss of growth control and the conversion of cell to a malignant state
- Accelerators of cell proliferation
- Lead to genetic instability
- Prevent cell from apoptosis
- Promote metastasis
- Act dominantly (only need mutant copy on one of the homologous
protoonco genes
- Have the potential to subvert cell own activities and push the cell towards the malignant state
- Encode proteins that have various function in cell normla activities
- Can be converted into the oncogenes:
- Mutation that will alter the properties of the gene product
- Multiple duplication -> gene amplification -> excess products
- Chromosome rearrangement ->altered gene expression
- After gain-of-function mutation->oncogene
mutations in RB genes that can lead to retinoblastoma
- RB gene is ex. of tumor suppressor gene
- Nonfamiliar ex of disease:
- Sporadic cases
- Person begins life w/ normal RB genes in zygote
- Retinoblastoma occurs only in whom a given retinal cell accumulates independent mutation in both alleles of the gene
- Familiar ex of disease:
- Ex inherited
- Abnormal allele in the zygote of the RB gene (usually present as a deletion)
- All cells of the retina have at least one unfunctional RB gene
- If the other gene is inactivated (ex. point mutation)->CA
Types of proteins encoded by proto-oncogenes:
Receptor for growth factors
Protein kinases and proteins that activate proteins kinass
Proteins that regulate cell cycle
Transcription factors
Proteins that modify chromatin
Metabolic enzymes
Proteins that inhibit apoptosis
Several families of oncogenes encode:
mutant forms of cytoplasmic protein kinases
Receptor protein kinases
GTP-binding proteins
Benign tumour and malignant tumour
- A benign tumour is composed of cancer cells that lack the capability to invade normal tissues or metastasize to distant cells
- a malignant tumour tends to metastasize.
Tumour-suppressor gene and oncogene
- A tumour-suppressor gene encodes proteins that restrain cell growth and prevents cells from becoming malignant,
- an oncogene encodes proteins that promote the loss of growth control and the conversion of a cell to a malignant state.
proto oncogene vs oncogene
- Proto-oncogenes are a variety of genes that have the potential to subvert the cells own activities, and push the cell toward a malignant state. They can be converted into oncogenes.
- An oncogene encodes proteins that promote the loss of growth control and the conversion of a cell to a malignant state.
The genes involved in carcinogenesis constitute a specific subset of the genome whose products are involved in distinct cellular activities. List three of these cellular activities.
Progression of a cell through the cell cycle
Adhesion of a cell to its neighbours
Apoptosis
Repair of DNA damage
Cell-to-cell communication
What are the mechanisms by which proto-oncogenes are converted to oncogenes?
- A gene mutation alters the structure and function of the encoded protein.
- A mutation in a nearby regulatory sequence alters the expression of the gene.
- A rearrangement of DNA brings a new DNA segment closer to the proto-oncogene, which results in an inappropriate transcription of the proto-oncogene by the promoter of the transposed gene.
