Cytoskeleton and Cell Structure

Cytoskeleton Overview

• The cytoskeleton consists of protein filaments within the cytoplasm.
• The cytoskeleton is highly dynamic and performs a number of important functions within eukaryotic cells
• The cytoskeleton is made up of Actin Filaments, Intermediate Filaments and Microtubules

Major Functions

• The cytoskeleton provides structural support, which helps to determine cell shape.
• The cytoskeleton provides internal organization by positioning organelles.
• The cytoskeleton facilitates in vesicle transport.
• Cell division relies on the cytoskeleton for chromosome segregation and overall cell division.
• The cytoskeleton contributes to large scale movements, like cell crawling and muscle contraction.

Microscopy Techniques

• Cytoskeletal filaments range from 7 nm to 25 nm in diameter.
• Light microscopes have a resolution limit of ~200 nm, constrained by the wavelength of visible light, making it impossible to resolve cytoskeletal filaments.
• Fluorescence microscopy uses a light microscope with the same resolution, however fluorescent labels are used to detect specific proteins, like cytoskeletal filaments.
• Transmission electron microscopes utilize beams of electrons to see structures with a short wavelength.
• This gives TEM microscopes a resolution of ~1 nm, capable of revealing detailed structures.

Immunofluorescence microscopy

• Immunofluorescence microscopy helps to specify the location of proteins.
• This type of microscopy is only compatible with cells that have been fixed, not living cells.
• This uses Primary and Secondary antibodies to bind to a specific protein
• Secondary antibodies are covalently tagged to a fluorescent marker.
• Fluorescence microscopes are used to excite the flourescent marker and visualise the light that has been emmitted.

Three Types of Filaments

• Actin filaments, with a diameter of ~7 nm, are one type of cytoskeletal filament.
• Intermediate filaments, with a diameter of ~10 nm, are another type of cytoskeletal filament.
• Microtubules, with a diameter of 25 nm, are a third type of cytoskeletal filament.
• All filaments are held together by noncovalent interactions.

Intermediate Filaments

• Intermediate filaments provide structural support, and have different types of IF proteins.
• Cytoplasmic IFs occur in animal cells subjected to mechanical stress and provide mechanical strength.
• Nuclear IFs include the nuclear lamina, which is a 2D meshwork consisting of lamins.

Cytoplasmic Intermediate Filaments

• Cytoplasmic IF proteins have a conserved α-helical central rod domain and N- and C-terminal domains that differ.
• Cytoplasmic IF pack together into rope-like filaments.
• 2 monomers form a coiled-coil dimer
• 2 dimers form staggered antiparallel tetramer
• 8 tetramers associate side by side and assemble into a filament
• Most interactions are noncovalent
• Cytoplasmic IF do not have filament polarity.
• Cytoplasmic IFs are tough, flexible, and have high tensile strength.
• Keratin filaments in epithelial cells create an interconnected network throughout the cytoplasm and are anchored at cell-cell junctions (desmosomes).
• Epithelium is a sheet of cells that cover external surfaces

Microtubules

• Microtubules' organizing function is present in all eukaryotes.
• Microtubules are involved in cell organization, including vesicle and organelle transport/positioning.
• Microtubules are involved in mitosis, and contribute to the structure of cells and structures like flagella.
• Microtubules are made of tubulin, forming long and stiff hollow tubes.
• They are inextensible (don't extend).

Microtubule Structure

• Microtubules are made of two related globular proteins called Alpha-tubulin and Beta-tubulin
• These form tubulin heterodimers that are bound to GTP.
• Alpha and Beta subunits are arranged for microtubule polarity, where the end (β) is different from the minus end (α).
• 13 parallel protofilaments make up a hollow tube.

Microtubule Protofilaments

• Protofilaments are bonded noncovalently
• Protofilament bonds are weaker than bonds within each protofilament.
• Microtubules can grow and disassemble at both ends, however this is faster at the plus end.

Microtubule Organising Centres (MTOC)

• Within the cell, microtubules grow out from MTOCs, for example the centrosome in animal cells.
• Ends are stabilised at the MTOC
• The plus ends of microtubules grow and shrink
• This continuous process needs remodeling
• These plus ends of microtubules can then grow or shrink
• MTOCs contribute to dynamic instability for microtubules
• The y-Tubulin Ring Complex (γ-TuRC) is an example of nucleation site.
• γ-TuRC is a protein complex of y-tubulin and accessory proteins, and ring of y-tubulin (gold) acts as an attachment site for αβ-tubulin dimers

Microtubule Dynamics

• The dynamic instability of microtubules is required in order to remodel.
• Microtubules in non-dividing animal cells radiate from one centrosome.
• In dividing animal cells, the centrosome duplicates to form two spindle poles which reorganise to form a bipolar mitotic spindle.
• Microtubule-associated proteins regulate the stability, dynamics, and organization of microtubules.

Microtubules & Transport

• Microtubules can be stabilized to prevent disassembly.
• Microtubules facilitate cargo transport in nerve cells (from the cell body to the axon terminal).
• Motor proteins on microtubules facilitate in axonal transport.

Motor Proteins and Intracellular Transport

• Kinesins move towards microtubules' plus end.
• Kinesin I transports organelles, vesicles, and macromolecules towards the axon terminus.
• Dyneins move towards microtubules' minus end.
• Cytoplasmic dynein transports worn-out mitochondria towards the cell body.

Organelle Positioning

• Microtubules are used to position organelles in cells.
• For example ER has kinesin-1 near to the nuclear envelope.
• Another example is the golgi which is positioned near to the centrosome by cytoplasmic dynein.

Actin Filaments

• Actin filaments are known as microfilaments and are present in all eukaryotes.
• Actin filaments are made of actin monomers, and are flexible but inextensible.
• Myosins are the motor proteins associated with actin filaments.
• Actin filaments contribute to stiff stable structures like microvilli or cell motility.

Actin Filaments Structure

• Actin filaments are usually 7nm in diameter
• Actin consists of:
• actin monomers
• noncovalent interactions
• two protofilaments twisted in a right direction
• Actin filament has polarity
• plus end is different from the minus end
• actin monomers are all in the same orientation in each protofillament
• growth is faster at the plus end of actin

Actin Monomers

• Free Monomers are bound to ATP
• Bound in the center of the protein
• Soon after the actin monomer has been added to the filament
• Actin hydrolyses ATP to ADP
• This reduces strength of binding between monomers in filament
• Rapid addition of Actin monomers
• faster than ATP hydrolysis in new ly added actin monomers
• Actin filament has an ATP cap

Actin Polymerisation in Test Tube

• This consists of
• Actin subunits and ATP added to a test tube
• In order to study actin filament polymeristaion
• Test tube also contains Nucleation - lag phase
  • Small oligomers form, but are unstable
• Includes Elongation - growth phase
• some oligomers become more stable, leads to rapid filament elongation (faster at plus end)
• Can also consist of stabilised actin -Steady state (equilibrium phase) • decrease in [actin subunits] • rate of subunit addition = rate of subunit dissassociation Treadmilling is the rate of the sub unit

Actin Filaments and Treadmilling

• This can consist of ATP as a building block of actin
• addition of actin monomers - polymerization
• actin monomers quickly hydrolyse to ADP
• Then will lose an actin monomer to be depolymerised
• this needs a continual supply of ATP building blocks
  • which can then produce the same size and looks stable
    • This means that the continues exchange of monomers at each end
      • Net addition at at the plus end
      • net loss at the minus end
      • this process moves throughout the filament
• Actin filaments must rapidly assemble at the leading edge
• this pushes the cell forward

Comparing Actin Filaments and Microtubules

• Actin Filaments
• Composed of ATP
• Have subunits that are connected after hydrolysis
• Have a smaller rate of synthesis compared to microtubules
• Mictotubules
  • Composed of GTP and a complex molecule chain
    • Sub Units Connected After Hydrolisis
• Both Have a good rate of synthesis

Cellular Structures & The Environment;

• This includes the next set of lessons and studies

Successfully Learning

• The student should understand the differences in microscope,
• the difference in different filament properties
• Describe the properties in the cytoskeleton and if filaments that form
• Describe how they perform - Dynamic instabilty
• Describe and Understand them.
• Understand and describe motor proteins and if actin filaments and how they move.