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Cell Motility and the Cytoskeleton: Part 1 Lecture NotesKey Wordscytoskeleton, actin, motility, force generation, Listeria, comet tail, neutrophil, keratocyte, Arp2/3 complex, filament dynamics, polymerization, nucleation, protrusion, branched network, Brownian ratchet Lecture Notes
There are three major types of cytoskeletal filaments in eukaryotic cells: actin filaments, microtubules and intermediate filaments The dynamic nature of filaments allows for rapid regulation of important cellular processes and the spatial distribution of filaments is essential for proper cellular organization. Actin filaments, concentrated beneath the plasma membrane, are involved in cell shape and motility. Microtubules direct intracellular traffic and the distribution of organelles as well as segregating chromosomes during mitosis. Intermediate filaments provide mechanical strength to structures (e.g. nuclei, cells and epithelial layers).
Bacteria also have structurally homologous cytoskeletal proteins Bacterial actin-like proteins are important in cell shape (e.g. MreB). Tubulin-like proteins are essential for binary fission (e.g. FtsZ). Intermediate filaments-like proteins are less common and are necessary for specific cell shapes (e.g. crescentin is required for the curved shape of Caulobacter crescentus).
Nucleation, polymerization and polarity of actin Cytoskeletal filaments are made of many individual subunits that have the inherent property of self-assembly, which allows the cell to form larger structures that can span throughout the entire cell body. Cytoskeletal filaments such as microtubules and actin filaments are typically made up of multiple protofilaments. Protofilaments are single strings of subunits that are joined end-to-end. Actin filaments are made of two protofilaments, while microtubules have about 13 protofilaments. Cytoskeletal filaments must be able to maintain an appropriate degree of stability while preserving their dynamic properties. For example, tight binding between adjacent subunits increases filament stability but is detrimental to filament dynamics, while weak binding between subunits increases subunit exchange but results in less stable filaments. Nucleation is the rate-limiting step of polymerization. Actin nucleation is stimulated by nucleating factors, such as the Arp2/3 complex, that promote the formation of a stable actin trimer that acts as the nucleus for polymerization. During actin polymerization, ATP-bound actin monomers (G-actin) add to existing filaments (F-actin). Nucleotide hydrolysis occurs shortly after the monomer assembles into the filament and produces ADP-F-actin, which is thermodynamically less stable than ATP-F-actin and therefore more likely to disassemble (i.e. ADP-F-actin favors filament depolymerization). Actin filaments have a fast-growing end called the plus end (or barbed end) and a slow-growing end called the minus end (or pointed end). The polarity of filaments results from the structural asymmetry of actin subunits and the head-to-tail assembly of subunits into filaments.
Actin bundles and branching Actin can form higher-order F-actin structures, such as filament bundles and dendritic branched networks, which have advantageous mechanical properties. Actin bundles are advantageous because they are stiffer than individual filaments, and therefore more resistant to bending and breaking, while allowing dynamical properties of individual filaments. In addition to nucleating actin filament growth de novo, the Arp2/3 complex can also bind to the side of existing actin filaments to nucleate the growth of a new filament that branches off the preexisting filament. This process results in the formation of branched webs of actin filaments. The role of branched actin networks in cellular motility will be discussed later.
Introduction to cellular motility Migration toward a chemical attractant is called chemotaxis. As an example, the neutrophil, shown in David Rogers’ movie, is chemotaxing up a gradient of chemotactic peptides that are produced by the bacterium. In a chemoattractant gradient, neutrophils become polarized and actively move toward higher concentrations of the chemoattractant. Chemotaxing amoebae look morphologically similar to neutrophils and they share many of the underlying molecular mechanisms that drive neutrophil motility. In addition, there are motile cells, such as cells involved in wound healing, that have distinct morphology and motility characteristics but preserve many of the molecular mechanisms important in neutrophil motility. This suggests that the molecular processes involved in cellular motility have been conserved over evolutionary time.
Steps in actin-based cell motility
In order for the cell to move, these events must be coordinated with the formation and disassembly of cell-surface adhesions. Typically, eukaryotic cells are on the order of tens of micrometers in size while the proteins these cells are built from are on the order of nanometers in size. This size discrepancy presents a problem of coordinating processes that are separated by distances that are many orders of magnitude larger than individual proteins. As a way to combat this problem, the cell uses filamentous structures that are assembled from many small subunits. In our discussion of cell motility, the cytoskeletal filaments of eukaryotic cells have three critical properties. First, filaments are able to assemble and disassemble rapidly, in order to change shape and orientation of a cell in response to changing signals from its environment. Second, filament assembly can function as a force generating mechanism that is important in protrusion of the leading edge. Third, cytoskeletal filaments can serve as directional tracks for molecular motor proteins (e.g. kinesin, dynein and myosin). This property of actin filaments is important for myosin-dependent retraction of the cell rear.
Cell Protrusion: leading edge moves forward The protrusion of the leading edge does not rely on preassembled structures. Rather, the cell must spatially regulate the assembly and disassembly of filaments. At the leading edge, actin polymerizes to form networks of filaments that assemble into parallel bundles and branched meshes. These networks of actin filaments exert a force that pushes against the plasma membrane and thus drives the protrusion of the leading edge. The process of assembly and disassembly of actin filaments is a highly dynamic process that is regulated by hundreds of actin-binding proteins. As a result, the activity and localization of actin-binding proteins must be carefully modulated to produce highly ordered outcomes that are observed during cellular motility. The dynamic nature of actin filaments is seen in filament turnover, where the average filament half-life is estimate to be approximately one minute.
Force generation during actin polymerization Actin polymerization can be modeled using the following binding reaction.
Where P1 is a single subunit, Pn is the filament length before subunit addition and Pn+1 is the filament length after subunit addition. kon is the on rate and koff is the off rate. In the cell, there is always an excess of monomers, thus the formation of filaments is energetically favorable. In the case of motility, the negative free energy produced during actin polymerization is coupled to drive the physical process of leading edge protrusion. This free energy is not free; remember, ATP is hydrolyzed after ATP-G-actin assembles into the filament.
Force generation by the addition of a single actin monomer The maximum force generated by the addition of a single monomer can be estimated using the equation
Where k is Boltzmann’s constant and T is the temperature in degrees Kelvin. At room temperature, kT ? 4.1 pN nm (picoNewton nanometers), this value represents the natural energy unit of a single molecule within a cell and is used as the natural energy scale in physical biology. The concentration of free actin monomers, defined as C, varies in different cell types but is typically on the order of a few tens of micromolar. Ccrit is the equilibrium constant for polymerization (Ccrit ? koff/kon ? 0.1 micromolar). (Note: Ccrit is different for ATP-G-actin and ADP-G-actin, and can also differ at the two structurally distinct ends of the actin filament; the number given is for ATP-G-actin at the plus end of the filament. The distance the load must move forward for new monomer incorporation is expressed as d (d ? 2.5 nanometers: 5 nanometer per subunit/2 protofilaments per actin filament). Then, plugging these values into the equation above: Fmax ? 5-10 pN. Putting this into perspective, the force generated from the assembly of a single subunit of actin is equivalent to the force generated from the power stroke of myosin and during a step by kinesin. Given this force, it is easy to imagine how the localized polymerization of many thousands of actin filaments at the leading edge of the cell would push the plasma membrane forward. The speed of network growth, generated by actin polymerization, is comparable with the rate of cell motility. Mathematical models predict the net polymerization rate to be on the order of one micrometer per second, which is similar to the speed of fast motile cells such as neutrophils.
The study of microbes can advance our understanding of basic cell biology There are a number of intracellular microbial pathogens (i.e. disease-causing microorganisms living within a host cell) that use host actin polymerization to propel themselves through the cytosol of the host cell and into neighboring cells, resulting in increased cell-to-cell spread of the microbe. Actin-based motility allows the microbe to infect and replicate within the host while actively avoiding the antibody-mediated arm of the host immune response. Examples of microbes displaying actin-based motility include Vaccinia virus and the bacteria Shigella and Listeria. In all instances, microbial surface proteins stimulate the Arp2/3 complex (either directly or indirectly), promoting actin polymerization at the microbial surface. Actin dynamics at the bacterial surface resemble the dynamics observed at the leading edge of a moving cell. Therefore, it is not surprising that the study of actin-based motility of intracellular microbes has increased our understanding of the molecular processes involved in cell motility.
Putting actin-based motility of Listeria into perspective Listeria is approximately 2 micrometers in length and travels through the cytosol at a speed of roughly 0.2 micrometers per second. Therefore, every ten seconds Listeria travels one cell length. The Ohio Class SSBN submarine has similarities to Listeria; both travel through a liquid medium, are similar in shape, and travel through their environments at a comparable relative speed. The submarine is 560 feet in length and travels at a speed of 30 feet per second. Therefore, in comparison, the relative speed of Listeria is approximately twice as fast as the submarine, which takes around 19 seconds to travel its length.
Biochemical events in actin-based motility of Listeria and Shigella
Questions to ponder How much force is generated by actin polymerization in vivo (efficiency)? How are multiple filaments coordinated to work together? How are actin forces coordinated with other cellular forces in both space and time? How are these processes regulated with the environment?
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