Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Abstract

The cell wall, a porous mesh-like structure, provides shape and physical protection for bacteria. At the atomic level, it is composed of peptidoglycan (PG), a polymer of stiff glycan strands cross-linked by short, flexible peptides. However, at the mesoscale, multiple models for the organization of PG have been put forth, distinguished by glycan strands parallel to the cell surface (the so-called "layered'' model) or perpendicular (the ''scaffold'' model). To test these models, and to resolve the mechanical properties of PG, we have built and simulated at an atomic scale patches of both Grampositive and negative cell walls in different organizations up to 50 nanometers in size. In the case of Gram-positive PG, molecular dynamics simulations of the layered model are found to elucidate the mechanisms behind a distinct curling effect observed in three-dimensional electron cryo-tomography images of fragmented cell walls. For Gram-negative PG, simulations of patches with different average-glycan-strand lengths reveal an anisotropic elasticity, in good agreement with atomic-force microscopy experiments. Insights from the simulations reveal how mesoscopic and macroscopic properties of a ubiquitous bacterial ultrastructure arise from its atomic-scale interactions and organization.

Myosin 18A Coassembles with Nonmuscle Myosin 2 to Form Mixed Bipolar Filaments

“…Consistently, we demonstrated in vitro that M18Ab reduces the size of NM2A filaments, possibly even preventing their formation when present in great excess. M18A could also regulate the mechanical output of filaments by reducing the number of force generating heads, thereby reducing processivity [17] (Figure 4A). It should be noted, however, that the ratio of M18A to NM2 in U2OS cells is 1:200 [15], arguing that M18A may not be present at sufficiently high levels in vivo to alter NM2 filament size (even in The addition of M18A to NM2 filaments may regulate filament size and mechanochemistry.…”

“…Labeled myosins were incubated in buffer A containing various modifications as indicted in the text. Flow cells (40 ml) were constructed as described previously [17]. Proteins at 5 nM were adsorbed directly onto glass coverslips, and the chamber was washed with 10-fold excess of buffer A containing 150 mM KCl and 50 mM DTT.…”

A Perspective on the Role of Myosins as Mechanosensors

“…The activities of both NMIIA and NMIIB have been suggested to be important for the formation and maturation of focal adhesions (50,51). Each of these myosins has different motile rates and duty ratios (59)(60)(61)(62)(63)(64)(65). Single NMIIA and NMIIB motors have been suggested to have a high-duty-ratio motor than can move processively along actin, but this assertion remains controversial (59)(60)(61)63,65,66).…”

“…The mechanical properties of NMIIC have not been measured. We assumed that NMIIC has a workingstroke displacement of 6 nm, similar to NMIIA (66), NMIIB (60), and other myosin-II isoforms (31,36,(66)(67)(68). We assumed that force slows the rate of ADP release and sliding velocity with a distance to the transition state of 3 nm in this isoform, similar to NMIIA (66), NMIIB (59), and smooth muscle myosin-II (31).…”

“…Each of these myosins has different motile rates and duty ratios (59)(60)(61)(62)(63)(64)(65). Single NMIIA and NMIIB motors have been suggested to have a high-duty-ratio motor than can move processively along actin, but this assertion remains controversial (59)(60)(61)63,65,66). Transient kinetic experiments suggest that NMIIA and NMIIB both show force-dependent changes in the rate of ADP release, where forces that resist the powerstroke slow the rate of ADP release, prolonging the attachment duration of actomyosin (65), but optical trapping experiments have shown conflicting results (59,60,66).…”