MreB, a major component of the bacterial cytoskeleton, exhibits high structural homology to its eukaryotic counterpart actin. Live cell microscopy studies suggest that MreB molecules organize into large filamentous spirals that support the cell membrane and play a key shape-determining function. However, the basic properties of MreB filament assembly remain unknown. Here, we studied the assembly of Thermotoga maritima MreB triggered by ATP in vitro and compared it to the wellstudied assembly of actin. These studies show that MreB filament ultrastructure and polymerization depend crucially on temperature as well as the ions present on solution. At the optimal growth temperature of T. maritima, MreB assembly proceeded much faster than that of actin, without nucleation (or nucleation is highly favorable and fast) and with little or no contribution from filament end-to-end annealing. MreB exhibited rates of ATP hydrolysis and phosphate release similar to that of F-actin, however, with a critical concentration of ϳ3 nM, which is ϳ100-fold lower than that of actin. Furthermore, MreB assembled into filamentous bundles that have the ability to spontaneously form ring-like structures without auxiliary proteins. These findings suggest that despite high structural homology, MreB and actin display significantly different assembly properties.

The conjugation of hydrophobic cytotoxic agents such as monomethyl auristatin E (MMAE) to the interchain sulfhydryl groups of monoclonal antibodies (Mabs) through a protease-labile linker generates a heterogeneous drug load distribution. The conjugation process can generate high-drug-load species that can affect the physical stability of antibody-drug conjugates (ADCs). In this study, the mechanism of physical instability of ADCs was investigated by formulating the ADC pool as well as isolated drug load species in high and low ionic strength buffers to understand the effect of ionic strength on the stability of drug-conjugated Mabs. The results showed that the presence of high ionic strength buffer led to time-dependent aggregate and fragment formation of ADCs, predominantly ADCs with high-drug-load species under stress conditions. In addition, differential scanning calorimetry (DSC) results confirmed that there is a direct correlation between thermal unfolding and drug payload and that specific changes in the DSC thermogram profiles can be assigned to modifications by MMAE.

The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins ␣-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than ␣-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.Monomeric filamin is a high molecular mass (250 kDa), 80-nm actin-binding protein that features an actin-binding domain in its N terminus and forms a V-shaped, flexible dimer (1). Filamin cross-links actin filaments into orthogonal networks below a threshold filamin concentration and bundles them above that threshold in vitro (2). Filamin isoforms are ubiquitously expressed in unicellular and multicellular organisms, eukaryotes, and prokaryotes (1). In cultured non-muscle adherent cells such as fibroblasts, filamin localizes to the cortical actin network, the base of cell membrane protrusions, and along stress fibers (3). In dividing cells, filamin is concentrated in the cleavage furrow, where it remains associated at the mid-body region until the completion of cell division (4). Expression of the dysfunctional human filamin-A causes the genetic disorder of ventricular heterotopia, presumably due to reduced neuronal migration to the cortex (5, 6). Filamin-A-null melanoma cells display plasma membrane blebbing (7), reduced migratory speed (8), and increased susceptibility to force-induced membrane leakage (9), all of which are phenotypes attributed to reduced stiffness of cortical actin. These observations, along with filamin's key role in actin organization, membrane stabilization, and the anchoring of transmembrane cell receptor proteins to the actin cytoskeleton, suggest that filamin has an important mechanical function (2).The mechanical function of filamin in vitro has ...

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.Prokaryotic actin homologues MreB/ParM/Mbl are, along with tubulin homologue FtsZ and intermediate-filament homologue crescentin, the major components of what appears to be an extended filamentous cytoskeleton in bacteria (28). Recent studies have demonstrated the importance of these proteins in bacterial functions (1,3,5,6,23,36). Fluorescence microscopy in vivo shows that MreB aggregates into a large filamentous spiral structure that lies underneath the cell membrane and spans the cell length (24). Several studies suggest an essential role for MreB in chromosome segregation (16,25), polar localization of proteins (15, 36), maintenance of cell shape, and resistance to external mechanical stresses. When MreB is depleted, the bacterial cell wall displays gross morphological defects (47): vibrioid-shaped Caulobacter crescentus cells become lemon-shaped (12), and rod-shaped Bacillus subtilis (5) and Escherichia coli cells (47) become rounded. Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog, Mbl (5); however, the mechanism by which MreB may provide mechanical support directly to the cell or indirectly by affecting peptidoglycan wall integrity remains unclear.In eukaryotic cells, cell stiffness is primarily provided by actin filaments, which organize into orthogonal arrays and ordered bundles that confer extraordinary elasticity to the cell (18). In physiological conditions, actin requires ATP or ADP to stabilize its folding and to polymerize (8). It has been reported that actin could polymerize in the presence of other nucleotides in vitro (31). Nevertheless, actin filament assembly and stability are highly favored in the presence of ATP and ADP (21,31,48). Yeast actin binds to and hydrolyzes GTP, but with much lower binding affinity and hydrolytic rate than ATP ...