Cytoskeleton II: Intermediate Filaments and Actin

• Wiley Plus assignment for chapter 7 and chapter 9 part 1 due this Friday
• Wiley Plus assignment for chapter 9 will be posted on this Friday
• That assignment is due March 28th (after spring break)
• Exam 2 is on March 25th
• Practice exam questions have been posted

Lecture 11- Cytoskeleton II

• The topics are
• Intermediate filament
• Actin Filament
• Motor Protein (part 1)
• Cytoskeletons have filamentous structure
• They form elaborate, interactive, and dynamic networks
• Cytoskeletons consist of polymers held together by weak noncovalent bonds
• Their weak bonds enable rapid assembly and disassembly
• Process of assembly/disassembly is dynamic

Intermediate Filaments (IF)

• Key functions of IF should be understood
• The molecules, distribution, and classification of IFs should be known
• You should know how IFs assemble and breakdown
• It is important to know the importance of IF in human biology
• Intermediate filaments have a rope-like, flexible shape
• IF are found only in animal cells
• These strong, flexible, ropelike fibers provide physical support to cells
• There are 70 different IF proteins
• There are five major IF classes based on cell type
• They are also determined by biochemical, genetic, and immunologic criteria
• Aggregation of neurofilaments is seen in neurodegenerative disorders
• Examples of diseases include ALS and Parkinson's
• Neurofilament aggregates may block axonal transport, leading to neuron death
• IFs are often interconnected to other cytoskeletons via thin, wispy cross-bridges
• Cross-bridges consist of the elongated protein called plectin
• Each plectin binds to IF at one end
• Depending on the isoform, plectin binds to either:
  • Another IF
  • Microfilament
  • Microtubule
• IFs provide structural support to nuclei
• They provide strength to tissues
• Desmin (muscle cell IF) mutation-related myopathy results in skeletal muscle weakness, cardiac arrhythmias, and eventual congestive heart failure
• Blistering disease is caused by mutations in intermediate filament proteins
• Epidermolysis bullosa(EBS) is a rare skin-blistering disease in humans( 50 in 1 million live births) – genetic autosomal disease
• Most EBS occurrences are due to point mutations (usually missense) in either Keratin 5 or Keratin 14 polypeptides
• Mechanical integrity of cells is one of the functions of intermediate filaments
• Another function is cell shape determination and Maintenance
• Similarly, another purpose is Cytoskeletal Cross Talk and Stability
• IF also act as signalling mechanisms and are involved in Adhesion
• They aid in cell motility
• IFs help with tethering and positioning organelles
• IF proteins are:
  • Heterogeneous
  • structually similar
• Examples of IF proteins Include
  • keratin
  • vimentin
  • desmin
  • lamin
  • nestin
  • Neurofilament
• Each monomer has a pair of globular terminal domains
• Domains are separated by a long alpha-helical region
• Alpha-helical region of the monomer becomes a coiled-coil dimer
• The dimer turns into a staggered tetramer
• The ropelike tetramer forms the basic building block of IF assembly
• Eight tetramers associate to form a unit length of the IF (about 60 nm).
• Unit lengths of filaments associate in an end-to-end fashion
• This builds forms a highly elongated intermediate filament.
• Tetrameric building blocks lack polarity
• Assembled filament also lacks polarity
• Subunits are incorporated in the middle
• Intermediate filament assembly does not require GTP.
• Assembly and disassembly are controlled primarily by phosphorylation and dephosphorylation

Actin filament Learning Objectives

• You should be able to:
  • Explain where F- and G-Actin are found in the cell, including the structures they contribute to
  • Compare and contrast F-Actin and Microtubule dynamics
  • Explain the actin treadmill model
  • Describe how actin filaments are regulated by various actin-binding proteins
  • Know the correlated prokaryotes cytoskeletal filaments
• Actin filaments are solid
• They often organize into a branching network
• Microfilaments are also known as actin filaments and F-actin
• They consist of a polarized polymer made of globular actin protein monomers (G-actin)
• An actin filament is a two-stranded structure
• This structure has two helical grooves running along its length
• Actin filaments can be organized into
  • Ordered arrays
  • Highly branched networks
  • Tightly anchored bundles
• The bundles can be found in hair cells (sensory cells for hearing and balance)
• F-actin is required for:
  • Cell motility/dynamics (cell migration during development, wound healing, protrusions, muscle construction, etc)
  • Transport
  • Structural support
• Elongation is much faster than nucleation (like MTs)
• There is a need for a "seed" or "nucleus" to start well
• Actin binding proteins facilitate
• Actin is an ATPase
• It hydrolyzes ATP to ADP after incorporation into the end of a growing actin filament
• An actin monomer (G-actin) binds a molecule of ATP before it is incorporated into a filament
• Subunits can be incorporated/released at either end
• Subunits tend to be added to the plus end and released from the minus end
• This process is actin treadmilling

Actin Binding Proteins

• Actin-binding proteins are important for quick changes in cell shape, and motility/dynamics
• They can be divided into categories based on their function in the cell:
  • Nucleating
  • Monomer-sequestering
  • End-blocking (capping)
  • Monomer-polymerizing
  • Actin filament depolymerizing
  • Cross-linking
  • Filament-severing
  • Membrane-binding
• Nucleation is energetically unfavorable
• Nucleating proteins are required
  • Examples are Arp2/3 complexes or Formins, including ActA ( in Listeria)
• Monomer sequestering proteins (thymosins) can alter G- to F-Actin equilibrium
• Capping proteins (e.g., tropomodulin in muscles) regulate and stabilize filament length
• Capping is acheived by binding to either end, resulting in a cap blocking both loss and gain of subunits
• examples or member-binding that is effective include:
• vinculin
• ERM family of actin-binding proteins
• spectrin, dystrophin (defects in dystrophin cuase muscular distrophy)
• filament-severing proteins, like cofilin, can break filaments into smaller pieces
• depolymerizing proteins, such as cofilin, will break down filaments

Prokaryotes

• Prokaryotes have cytoskeletal filaments
• Division Eukaryotes: use Tubulin Prokaryotes: use FtsZ
• Polarity Eukaryotes: use Actin Prokaryotes: use MreB
• Shape Eukaryotes: use Intermediate filaments Prokaryotes: use CreS

Motor Proteins

• Microtubule-associated motor Proteins are kinesins and dyneins
• Actin filament-associated motor Proteins are myosins
• Each of the motor proteins are coded by multiple genes
• Motor proteins utilise ATP to move in opposite directions along microtubules (MTs)
• Motor proteins move uni-directionally along their cytoskeletal track in a stepwise manner.
• Motor proteins move cargo
• The routes they take travel along microtubule "tracks"
• Kinesin is a plus-end directed microtubular motor
• Kinesin carries ~45 different kinesin-related proteins *(KRPs)
• *KRPs are tetramers of two identical heavy and two identical light chains
• Heads "step" along protofilament, 1 tubulin dimer at a time towards + end--> moves in a “hand-over-hand" mechanism
• ATP Is dependent in the globular heads
• Process facilitates the globular heads to bind microtubules and act as ATP-hydrolyzing, force-generating engines
• Processive motions make it possible to walk along a microtubule for considerable distances without falling off.
• Kinesins tend to move organelles (mitochondria, peroxisomes) and vesicles outward
• Dynein is a minus-end directed microtubular motor
• It is implicated in cilia and flagella movement
• later found in all animal cells (cytoplasmic)
• The stalk binds Microtubules
• Dynactin proteins binds cargo
• It moves organelles, vesicles, particles, positions Golgi, centrosomes, meiotic spindle (required for cell division)
• Motor proteins maintain cell organization
• Flagella/cilia move
• Examples of sperm motility that can be viewed include( mouse_ _http://www.youtube.com/watch?v=IEdb3wTMSBo - human: _http://www.youtube.com/watch?v=vvnEsOaKxuw&feature -related _http://www.youtube.com/watch?v=-. cilia - Flagella and cilia are examples of locomotion
• Cilia are microtubule-rich structures that can be motile or non-motile
• Multicellular organisms use cilia to move fluid/particles
• Primary cilia are often non-motile but can sense the local environment
• Flagella also are microtubule-rich structures that beat or wave
• Protozoans, algae, and sperm use flagella for movement
• Power stroke is the "push", but recovery stroke swings back into position
• Dynein is responsible for generating bending of the bending motion of cilia and flagella
• Cilia and flagella grow via intraflagellar transport
• Intraflagellar transport delivers process through which material is transported along the cilium and flagella
• IFT proteins assemble into a protein complex called the IFT particle
• These particles form linear arrays called IFT trains
• Similarly, they carry cargo proteins
• Such as tubulin out to the tip for assembly
• Inhibition of IFT prevents assembly of cilia and flagella.
• Bardet-Biedl syndrome -ciliopathy: mutations affect cilia/basal bodies
• This can result in polycystic kidney disease, situs inversus, retinal degeneration, and smell loss