Bio202 Lecture 11: The Cytoskeleton Part 1 (Overview) PDF

Lecture 11: The Cytoskeleton Part 1 (Overview) Today’s agenda: Overview of cytoskeletal filament systems: Intermediate filaments, Actin filaments, Microtubules ▪ subunits, filaments, motors ▪ goal is compare and contrast the major properties (will go more in depth in the next...

Lecture 11: The Cytoskeleton Part 1 (Overview) Today’s agenda: Overview of cytoskeletal filament systems: Intermediate filaments, Actin filaments, Microtubules ▪ subunits, filaments, motors ▪ goal is compare and contrast the major properties (will go more in depth in the next two lectures) Figure 17-1  cytoskeletons organized networks of polymers within cells “Scaffold-like” appearance of cytoskeletal networks Cytoskeletal networks can provide mechanical strength Eiffel Tower Support Actin filaments (imaged by EM) Cytoskeletal networks can also be dynamic  Three cytoskeletons in eukaryotic cells ▪ Each made up of subunits that combine to form polymers/filaments ▪ Polymers form through non-covalent interactions (reversible protein-protein interactions) Figure 17-2  Intermediate filaments Red = cell membrane Blue = intermediate filament ▪ Identified in muscle cells protein (keratin) ▪ Named because they had an “intermediate” diameter between actin filaments and myosin filaments (will discuss muscle and myosin later in this lecture) ▪ Made of fibrous proteins (different kinds in different tissues) ▪ Flexible and have high tensile strength (can withstand stress) Figure 17-2, 17-3  Intermediate filaments – the subunit Figure 17-4 ▪ Two dimers arranged in antiparallel fashion ▪ Dimers stacked on top of each other, but slightly offset ▪ N-termini stick out at each end Monomer (1) dimer (2) tetramer (4) IF subunit Intermediate filaments – the polymer Monomer = single copy of the protein Monomers form parallel dimers Dimers form antiparallel tetramers (This tetramer is the subunit for intermediate filaments) 8 tetramers associate laterally (side-to-side) Groups of 8 tetramers associate end-to-end Figure 17-4 Intermediate filaments have particularly strong lateral interactions Figure 17-4 ▪ Subunits are long and pack together along their lengths ▪ Many lateral interactions along the lengths of the subunits ▪ Gives the filaments strong rope-like properties ▪ Filaments provide structural support to cells and enable cells to withstand mechanical stress  Types of intermediate filaments Will use keratin Will also discuss as an example of nuclear lamins a cytoplasmic IF Figure 17-5 ▪ Intermediate filaments found in the cytoplasm of most (not all) types of animal cells ▪ Type of cytoplasmic intermediate filament varies by cell type ▪ Nuclear lamins found in all animal cells  Cytoplasmic intermediate filaments Example: Keratin filaments Keratin filaments distribute stress when skin is stretched ▪ Filaments within the cytoplasm of cells (not the nucleus) Figure 17-3, 17-5 ▪ Anchored to the plasma membrane to provide structure to the cells ▪ Filaments linked to sites of connection with neighboring cells (these connection sites are called desmosomes; will discuss in lecture 19) ▪ Linking cells mechanically couples them, giving structure to sheets of cells  Cytoplasmic intermediate filaments WT keratin: Orderly array of cells Mutant keratin: Tissue ruptures Epidermis bullosa simplex – a rare genetic disease associated with mutations in keratin. As a result, the skin is highly vulnerable to mechanical injury and even gentle pressure can cause skin to blister. Figure 17-6 Figure 17-5, 17-7 Nuclear intermediate filaments Nuclear lamins support and strengthen the nuclear membrane EM of the nuclear lamina: (view from inside the nucleus)  Nuclear intermediate filaments Nuclear lamins support and strengthen the nuclear membrane Progeria – a premature aging disease caused by defects in a particular nuclear lamin protein Normal lamin: Mutant lamin: Normal nucleus Irregular shape Figure 17-8 Take home: Intermediate filament proteins give support to the cells themselves (cytoplasmic IFs) or to the nucleus (nuclear lamins)  Actin filaments ▪ Present in all eukaryotes ▪ Actin filaments are highly dynamic – subunits can be added/removed from the ends Figure 17-2, 17-29  Actin filaments – the subunit Subunit = a single copy of the protein actin Structure: Simplified diagram: (Image from Alberts, Figure 17-30 Molecular Biology of the Cell)  Actin filaments – the polymer ▪ The subunit is a single monomer of actin ▪ Monomers assemble into a two-stranded helix Will discuss actin and its functions more in lecture 12 Figure 17-30  Microtubules ▪ Present in all eukaryotes ▪ Hollow cylinders made of tubulin heterodimers ▪ Highly dynamic Figure 17-2, 17-11  Microtubules - the subunit ▪ Each subunit is a dimer of -tubulin and -tubulin (two different but highly homologous proteins that form a heterodimer) ▪ Heterodimer formed through non-covalent protein-protein interactions, but the two proteins are tightly bound and never come apart Structure: Simplified diagram: Figure 17-12 (Image from Alberts, Molecular Biology of the Cell)  Microtubules – the polymer Cross-section view ▪ Each subunit is a heterodimer of -tubulin and -tubulin ▪  heterodimers assemble into protofilaments ▪ Lateral interactions between protofilaments form tube (13 protofilaments in tube) Will discuss microtubules and their functions more in lecture 13 Figure 17-12  Summary: The three cytoskeletal networks Figure 17-2 ▪ Filaments look different ▪ Built from different types of subunits Which of these filaments have polarity?  Filaments have polarity if the subunits are asymmetric The subunits: Antiparallel tetramer Symmetric Asymmetric Asymmetric The two ends of Dimer has 𝛂-tubulin Although the actin the subunit look on one end and β- subunit is a the same tubulin on the other monomer, the two sides of the protein look different Figure 17-2 Actin filaments and microtubules have polarity while IFs are non-polar The subunits: Antiparallel tetramer Symmetric Asymmetric Asymmetric The filaments: Non-polar Polar Polar Figure 17-2 Actin and microtubules have “plus” and “minus” ends ▪ Because the subunits are asymmetric, the two ends of the filament look different ▪ Note: “Plus” and “minus” refer to dynamic properties, not charge Figure 17-12 Figure 17-30 How do we know that the ends have different dynamic properties? Experiment: ▪ take short “seed” (pre-formed actin filament) ▪ add actin to polymerize off the ends of the seed End of seed End of seed Short actin seed stub growing off this end (pre-formed actin Long actin filament, decorated Slower with a protein to filament growing growing label and stabilize it) off this end Faster growing Conclusion: The two ends of the actin filament are different ▪ Faster growing end designated “plus” ▪ Slower growing end designated “minus” Short actin seed stub growing off this end (pre-formed actin Long actin filament, decorated Slower with a protein to filament growing growing label and stabilize it) off this end Faster growing Microtubules also have a fast growing “plus” end and a slow growing “minus” end ▪ If you did a similar “seed” experiment to the one we talked about for actin, the microtubule plus end would grow longer Minus Plus end end - MT “seed” + ▪ Pre-formed microtubule “seed” depicted in dark green ▪ Additional subunits added on during the experiment depicted in light green  Why would you want filaments to have polarity? One reason: ▪ Actin and microtubules can serve as “tracks” within the cell, for the transport of other things (organelles, vesicles, etc.) ▪ There are motor proteins that can walk along these tracks ▪ Each class of motors walks in a specific direction, which allows directed transport of components to specific locations in the cell Example of motor transport: ▪ This clip from the Inner Life of the Cell movie shows a motor (kinesin) walking on microtubules  Microtubule motor proteins: dynein and kinesin ▪ “Head” domain binds to microtubule ▪ “Tail” domain links to cargo ▪ Many classes of motors are dimers, so have two heads that “walk” along the MT Dynein: Kinesins: Walks to MT Many families, minus ends most walk to MT plus ends Figure 17-20  Motors “walk” along cytoskeletal filaments Figure 17-24 ▪ Walking is driven by ATP binding and hydrolysis in the two head domains (causes conformational changes)  Actin motor proteins: Myosins ▪ ATP binding, hydrolysis, and dissociation leads to conformational changes that allow the motors to “walk” along actin filaments (same principle as kinesins/dynein) ▪ There are multiple classes of myosin motors that perform different cellular functions ▪ Most myosins walk to actin plus ends Note: There are no known motors that walk on intermediate filaments (those filaments are symmetric and would not allow directed transport) (Note: you do not have to memorize all of these types) Motors can transport cellular components (e.g. vesicles/organelles) ▪ Actin/microtubules can act as tracks for motors to walk on ▪ Filament remains in one place, motor transports things along it vesicle Motor walks (-) end Stationary filament (+) end vesicle (-) end Stationary filament (+) end But motors can also slide filaments to move them within the cell ▪ In these cases, the motor remains in place ▪ The motor walks on the filament ▪ This causes the filament to slide relative to the motor (-) end (+) end Motor walks Anchor point Note: If motor walks to the filament (-) end (+) end plus end, this pushes the minus end forward (minus end is “leading”) Filament slides Anchor point  Experimental demonstration that motors can slide filaments: gliding filament assay ▪ Motors attached to a microscope slide, with their heads up ▪ Filaments added on top and ATP added to activate the motors – motors walk on the filaments and push them around the microscope slide Motor walks to plus end, this pushes Filaments (artificially colored so you the minus end forward (minus end is can track them) gliding on top of a slide: “leading”) Motors attached to slide, with heads pointed up Figure 17-23 The job of some motors is to slide filaments within the cell Example: Myosin II ▪ Myosin II is a dimer ▪ Heads walk on actin ▪ Coiled coil tail ▪ Multiple motors can assemble together to form a bipolar filament (“bipolar” since heads pointing in opposite directions) ▪ These bipolar filaments can slide actin filaments Figure 17-38 Bipolar Myosin II filaments can form contractile structures Bipolar myosin II filaments can slide actin filaments, forming contractile structures in cells: ▪ Heads on each side walk to plus ends ▪ This walking slides the two filaments relative to each other (top filament slides left, bottom filament slides right) Figure 17-39  Myosin II filaments drive muscle contraction Sarcomere = a contractile unit of actin and myosin in muscle Figure 17-42 ▪ Muscles contract by a “sliding filament” mechanism ▪ Contraction is driven by myosin heads walking to the plus ends of actin filaments, shortening the sarcomere Next lecture: The actin cytoskeleton in depth ▪ General principles of polymerization ▪ Regulation of actin in cells ▪ Functions of the actin cytoskeleton