Lecture 15 & 16 Cytoskeleton and cell motility Flashcards
What is a cytoskeleton?
A network of interconnected filaments and tubules
- Polymers
- Dynamic
What is the function of the cytoskeleton?
- Cell structure and mechanics
- Force generation – motility
- Intracellular transport
- Cell division
What are the 3 major structural elements that make up the cytoskeleton?
Ø Microtubules
Ø Microfilaments
Ø Intermediate filaments
What are microtublues?
- make up of heterodimers of alpha and beta tubulin proteins, which wrap around eachother to form a tubular shape.
- it has (+) and (-) ends
- are the largest of the cytoskeletal elements of a cell
What are microfilaments?
- made up of globular proteins (G - actin monomers) to produce two interwinded F - actins filaments
- has (+) and (-) end
- the smallest of the cytoskeletal filaments
What are intermediate filaments?
- intermediate in size bwteen microtubules anf microfilaments
- alot of variation between them
- made up of individual protien dimers end to end and side to side, then build up
What are the two types of microtubules? Function?
- Cytoplasmic microtubules: found throughout cytoplasm, used for regulation of cell structure, vesical transport (axons) and divison (formation of mitotic spindles).
- Axonemal microtubules (organised and stable): found in flagella, cilia and basal bodies; used for cell motility and signalling hub (secondary use)
What are the building blocks of microtubules?
What is microtubule polymerisation and assemnly process?
- highly regulated and dymanic process
3 steps:
- Nucleation
- Elongation
- Platueau (treadmilling)
How are microtubules formed?
- Nucleation: tubules start to get together and assemble small stable structure.
- Elongation: radpidly elongate to form a long tubule
- Plateau: stop growing (steady state)
Why?
- the rate of addition is proportional to the concentration of free tubulin
Microtubule growth is dependent on what?
concentration
• Critical concentration – is the tubulin concentration at which MT assembly is exactly balanced with disassembly
What is the effect that the MT growth is polarised?
High affinity for + end (add more to this end)
Low affinity for - end (add less to this end)
What happened at the steady state (treadmilling)?
What is dymanic instability?
microtubules can switch between phases of rapid growth, rapid shrinkage and dissembly.
The model: one population of MTs grows by polymerization at the plus ends whereas another population shrinks by depolymerization
Individual MTs can go through periods of growth and shrinkage: microtubule catastrophe - a switch from growth to shrinkage microtubule rescue - a sudden switch back to growth phase
What causes dynamic instability?
Due to the GTP cap
At low tubulin concentrations:
- GTP hydrolyzes, which causes its own depletion of the GTP cap, making it unstable, leading to catastrophe
How are the MT organised?
Microtubules originate from a microtubule-organizing center (MTOC), aka the centrosome
How is MT growth and stability regulated?
• Microtubule-stabilizing/bundling proteins:
- tau
MAP2
+–TIP proteins
• Microtubule-destabilizing/severing proteins:
- Stathmin/Op18
Catastrophins
katanins
What are examples of MT inhibitors?
- Colchicine - promotes MT disassembly
- Nocodazole - inhibits MT assembly
anitmitotic drugs: interferes with spindle assembly, inhibits cell division and useful for cancer treatement
What is the role of microfilaments?
– Muscle contraction
– Intracellular tension and cell shape
– Cell migration: lamellar and amoeboid movement
– Cytoplasmic transport
What is actin?
is the monomeric building block in microfilaments
- 42 kDa protein
- Abundant in all eukaryotic cells
- Polymerises into filaments
- Binds ATP/ADP
Multiple types:
– α-actin (muscle specific)
– β-actin and γ-actin (all cells)
G-Actin monomers polymerize into ____ microfilaments. How does this work?
G-Actin monomers polymerize into F-Actin microfilaments.
- form two coiled filaments bound by ATP or ADP.
- they bind when ATP is introduced, having a higher affinity to (+) end
- ATP hydrolizes into ADP, losing phsphate group becoming less stable and therefore more prone to dissembly on the (-) end
Actin microfilaments interacte with ___.
Actin microfilaments interacte with myosin.
- myosin proteins will bind to the actin monomers, with a particular orientation depending on if it is on the (+) or (-) end, having a barbed structure.
- myosin are motor protiens which pull on actin microfilaments and creating force.
Actin polymerisation is regulated by ____ .
Actin polymerisation is regulated by many diff proteins.
What are the actin binding proteins? (5 points)
- Profilin - binds ATP actin and promotes polymerisation
- Arp2/3 – promotes nucleation and branching
- Formins – Bind actin filaments and promote elongation
- Capping proteins - bind the ends of a filament; prevent further loss/addition of subunits e.g. CapZ; tropomodulins
- ADF/cofilin - binds G-actin and F-actin (also severs filaments)
What is Arp2/3?
- most important regualtors of actin growth and assembly
- binds actin monomers and holds them in a certian way so other monomers and add onto the end
- its other end can bind to existing actin filaments
Why is the assembly of actin so complicated?
Because of how diverse F-actin structures and functions are
What are the properties of intermediate filaments?
- the most stable
- the most diverse
- the least soluble
- are not polarized
- 6 different classes of intermediate filaments
Examples of IFs?
- Keratin
- Vimentin
- Lamin
- Desmin
How are Intermediate filaments assembled?
- composed of dimers, which form with other dimers to produce a tetradimer and then link end to end and side to side to make a long chain (proto filament), which bundle in groups of 8 to produce an intermediate filament.
How do cytoskeletal elements integrate?
creates a cohesive mechanical system
The cytoskeleton physically links to the external environment by adhesion receptors:
– Integrin receptors link cells to the extracellular matrix (e.g. collagen)
– Cadherin receptors link cells to other cells
What are integrin receptors?
- transmembrane receptors, with both an extracellular domain and intracellular domain
- always expressed in a pair (alpha and beta subunit)
- complex that binds ot actin filaments, which binds extracellular proteins to receptors to cell cytoskeleton
What is hemi-desmosome?
- dense, stable cluster of integrins that link to intermediate filaments
- also a complex that binds to intermediate filaments, which binds extracellular proteins to receptors to cell cytoskeleton
What are cell-cell adhesions?
Cadherin receptors: Bind cadherins on adjacent cells (homotypic interaction)
Two classes:
- Adherens junctions link to F - actin microfilaments
- Desmosomes link to intermediate filaments
What are motile systems?
A system of motor proteins and structural proteins that facilitate movement and transport
- Cytoskeleton provides a scaffold for motor proteins or mechanoenzymes
- Motility occurs at the tissue, cellular, and subcellular levels
– Muscle contraction, cell migration, and intracellular transport
Examples of cell motility?
- Development: border cell migration
- Intracellular: chromosome separation
- Keratinocyte Wound Healing
What are the 2 Eukaryotic motility systems?
2 major systems:
- Interactions between motor proteins and microtubules – e.g., fast axonal transport in neurons, or the sliding of MTs in cilia and flagella
- Interactions between actin and members of the myosin motor proteins – e.g., muscle contraction
What is MT-based movement?
MTs - provide a rigid set of tracks for transport (e.g. variety of organelles and vesicles)
- Dynein “inbound” - traffic toward the minus ends
- Kinesins “outbound” – traffic toward the plus ends
What are kinesins?
Kinesins consist of three parts:
- a globular head region that attaches to MTs
- a coiled helical region
- a light-chain region (attaches the kinesins to other proteins or organelles)
How do kinesins move?
Kinesins “walk” along microtubules.
- motor proteins exist as a dimer (each have a heavier and lighter domains)
- one will be attached to the microtubule, and thtrough the conversion of ATP to ADP, the release of energy will cause a conformational change - the other foot will attach to the microtubule releasing the other.
How do dynein motor proteins move?
- heavy chains interaction with the microtubule and light chain interacting with the cargo membrane
- through the action of ATP turning into ADP they move along microtubule
Cilia and Flagella require ____ for movements.
Cilia and Flagella require microtubules for movements.
What is the strucutre of cilia and flagella?
The Axoneme: shared structure
9+2 structure: 9 outer doublets and 2 inner MTs
Separated by 9 dynein molecules
Interconnected MTs and motor proteins
How does dynein generate the bending of cilia and flagella?
The sliding-microtubule model
The driving force for MT sliding is provided by ATP hydrolysis of dynein.
What is Actin-based cell motility?
- Myosins - ATP-dependent motors
- Tetramer - two heavy chains and two light chains
- 24 known types of myosins
- Function in a wide range of cellular events:
– Muscle contraction
– Cell movement
– Endocytosis
What is Actin-based cell movement, regarding the myosins?
- The globular head binds actin
- Most move toward the plusend but myosin (VI is an exception)
- common structure
How do myosins generate force and pull on actin?
- They use ATP hydrolysis to cause actin filaments to slide past myosin molecules
- Myosin II is an efficient motor that “walks” along actin (like kinesin)
How do Filament-based movements work in muscles?
- Skeletal muscles are responsible for voluntary movement
- A muscle consists of parallel muscle fibers
Each fiber is a long, thin, highly specialized, multinucleate cell
What is the structure of skeletal muscle cells?
What are Thick filaments?
- Consist of hundreds of molecules of myosin
- The myosin is arranged in staggered fashion
- Protruding heads of myosin molecules contact the adjacent thin filaments, forming cross-bridges
What are Thin filaments (aka F-actin)?
- Interdigitate with the thick filaments
- Contain three proteins: F-actin, intertwined with tropomyosin and troponin
How do Muscles contract?
Calcium dependent
- Complex of troponin molecules bound to F-actin MT, bound in a way which prevents Myosin from binding and pulling on it (resting condition
- However, when there is neural stimulation it increases calcium stores which bind to troponin which causes a conformational change that allows myosin to bind
- Important for control of muscle contraction
How does Actin-based motility work in non-muscle cells?
• Microfilaments – required for the movement of most animal cells • Cell crawling involves: -
extension of a protrusion
- attachment to substrate
- generation of contraction via myosin motors
What are Extending protrusions?
To crawl, cells extend protrusions
Filopodia: Thin finger-like projections
Lamellapodia: Large flattened projection
What happens during cell migration? (cell crawling and adhesion)
- Actin polymerisation drives protrusion of lamellapodia
- New sites of attachment form at the front of a cell – ‘molecular clutch’
- Acto-myosin contractility pulls rear of the cell
- Focal adhesion contacts at the rear detach
What is Amoeboid movement?
Amoebas and white blood cells exhibit a type of migration called amoeboid movement, which is accompanied by protrusions of a pseudopodia (‘flase foot’)
- depolimerisation of actin at the front, contraction and squeezing through a space.
What is Transendothelial Migration?
- improtant movement for immune cells and often found in pathogenic processes
How does Actin based motility work in Endocytosis?
Actin polymerisation and contractility facilitates endocytosis of clathrin coated pits
How does Actin based motility work in Cytokinesis?
Analogous systems within bacterial community, Bacterial cytoskeletal systems - what are the proteins present?
FtsZ Protein – is a prokaryotic homologue to the eukaryotic protein tubulin – division regulation
Crescentin – is a bacterial relative of the intermediate filaments – cell shape
MreB protein – is similar to actin microfilaments – cell shape
Summary:
- Motile systems consist of motor proteins and cytoskeletal scaffolds and facilitate movement and force generation.
- Kinesin and dynein are motor proteins that walk along MTs, transport vesicles, and move axonemal structures.
- Myosins pull on actin microfilaments to generate tension – muscle contraction and cell motility.
- Cells migrate through coordinated crawling and ameoboid movements.
- Prokaryotic cells have analogous cytoskeletal structures to eukaryotes.
