Archaea play indispensable roles in global biogeochemical cycles, yet many crucial cellular processes, including cell-shape determination, are poorly understood. Haloferax volcanii , a model haloarchaeon, forms rods and disks, depending on growth conditions. Here, we used a combination of iterative proteomics, genetics, and live-cell imaging to identify mutants that only form rods or disks. We compared the proteomes of the mutants with wild-type cells across growth phases, thereby distinguishing between protein abundance changes specific to cell shape and those related to growth phases. The results identified a diverse set of proteins, including predicted transporters, transducers, signaling components, and transcriptional regulators, as important for cell-shape determination. Through phenotypic characterization of deletion strains, we established that rod-determining factor A (RdfA) and disk-determining factor A (DdfA) are required for the formation of rods and disks, respectively. We also identified structural proteins, including an actin homolog that plays a role in disk-shape morphogenesis, which we named volactin. Using live-cell imaging, we determined volactin’s cellular localization and showed its dynamic polymerization and depolymerization. Our results provide insights into archaeal cell-shape determination, with possible implications for understanding the evolution of cell morphology regulation across domains.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation, and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than like that of slow anchoring myosin-1s found on endosomal membranes. We therefore propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells. Summary:Pedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5's role in clathrin-mediated endocytosis are discussed.

It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions.

Myosin VI physiologically functions as transporter and anchor in a cell.Recent single molecule high accuracy nano imaging (Nishikawa et al., 2010) revealed myosin VI has inchworm-like steps and hand-over-hand steps.These diverse stepping mode may relate to the switch between transporter and anchor, however, there's no evidence for that.Here, we examined the relashionship by a direct observation of stepping dynamics under tension, because tension triggers the switching between transporter and anchor.We achieved FIONA under tension by constructing nano sized spring using DNA nanotechnology (DNA origami).We found, during anchoring, myosin VI does not only prolong dwell time but repeats inchworm-like step and back steps to keep the anchoring state.

Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those “not seen.” Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.

Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability – as is evident in a disease such as Duchenne Muscular Dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we express and physically characterize dystrophin ‘nano’-constructs based on multi-exon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) which can detect tryptic peptides from 53 of dystrophin’s 79 exons; for equivalent information from immunodetection, 53 different high-specificity antibodies would be required. Folding predictions for the nano-constructs reveal novel helical bundle domains arising out of exon-deleted ‘linkers’, while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an Atomic Force Microscope (AFM) nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multi-exon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon-skipped designs.

Among the myosin family, while other myosins move toward the barbed(+) end, myosin VI moves toward the pointed (-) end of the actin filament. This unique feature allows myosin VI to fulfill multiple cellular processes such as clathrin dependant endocytosis when bound to Dab2 or clathrin independant when linked to GiPC, vesicules transport to the Golgi and the perinuclear region, vesicules transition from early endosome to late endosome, AMPA receptor trafficking when bound to SAP-97. Our working model for how myosin VI function in a cell is that the full-length myosin VI molecule primarily exists as a monomer, and folded to form intramolecular interactions involving the cargo-binding domain that block potential dimerization sites. This is consistent with small-angle X-ray scattering data obtained by Spink et al.. Binding to monomeric cargo adaptors leads to an unfolding of the monomers, exposing potential dimerization sites. The unfolded monomers can then be held in close proximity, either via tethering by the adaptor protein and/or via as yet unidentified cargo-binding domain interactions. Alternatively, binding to a dimeric cargo adaptor protein leads to simultaneous unfolding and close opposition of the cargo-binding domains. This distal tethering of two cargo-binding domains allows internal dimerization (likely via coiled coil) to occur at the proximal end of the medial tail, and may include part of the last helix of the three-helix bundle. This internal dimerization causes the three-helix bundle, formerly known as the proximal tail, to unfold, forming an extension of the myosin VI lever arm.Although it has been a controversy whether myosin VI exists as a monomer or dimer, our data and others support the model in which myosin VI exists as monomer in cell but upon binding to cargo binding proteins allows its dimerization and ultimately fulfills its function in cell.

Myosin VI is an actin-based molecular motor with a large step size despite its short lever arm. We recently showed that the medial tail domain of myosin VI, likely a ∼70-residue long alpha-helix, is a dimerization region when two monomers of myosin VI are brought into close proximity. This proximity mimics the effect of cargo binding onto myosin VI monomers. The medial tail domain has peculiar characteristics. Although there are a few hydrophobic residues in the lower amino acid number region, it has a distinct motif alternating between four positively charged residues and four negatively charged residues. Single-molecule experiments show that a mutant construct of the myosin VI medial tail domain, designed to disrupt the hydrophobic interactions of myosin VI, can still dimerize, therefore ruling out the hypothesis that hydrophobic interaction is the only dimerization mechanism of the medial tail domain. Additionally, it was observed that increasing ionic strength reduces the percentage of myosin VI dimerization. To unveil the molecular mechanisms involved in the dimerization of the medial tail domain, we employed three different molecular dynamics (MD) methodologies, namely: 1) coarse-grained MD facilitating microsecond timescale sampling; 2) the flexible fitting method enabled reversion of coarse-grained to all-atom descriptions; 3) all-atom equilibrium MD. The coarse-grained MD simulation showed the medial-tail domain dimerized, and the all-atom flexible-fitting method identified interactions between two medial-tail domain helices. We used the all-atom equilibrium MD simulation to make sure that the interactions involved in dimerization were stable over time. This set of simulations suggests that an array of salt bridges between positive and negative residues participates in the dimerization process. These results could have an important implication on myosin VI dimerization.

Force-bearing linkages between the cytoskeleton and extracellular matrix are clearly important to normal cell viability-as is evident in a disease such as Duchenne muscular dystrophy (DMD) which arises in the absence of the linkage protein dystrophin. Therapeutic approaches to DMD include antisense-mediated skipping of exons to delete nonsense mutations while maintaining reading frame, but the structure and stability of the resulting proteins are generally unclear. Here we use mass spectrometry to detect most dystrophin exons, and we express and physically characterize dystrophin "nano"-constructs based on multiexon deletions that might find use in a large percentage of DMD patients. The primary structure challenge is addressed first with liquid chromatography tandem mass spectrometry (LC-MS/MS) which can detect tryptic peptides from 53 of dystrophin's 79 exons; equivalent information from immunodetection would require 53 different high-specificity antibodies. Folding predictions for the nano-constructs reveal novel helical bundle domains that arise out of exon-deleted "linkers," while secondary structure studies confirm high helicity and also melting temperatures well above physiological. Extensional forces with an atomic force microscope nonetheless unfold the constructs, and the ensemble of unfolding trajectories reveal the number of folded domains, proving consistent with structure predictions. A mechanical cooperativity parameter for unfolding of tandem domains is also introduced as the best predictor of a multiexon deletion that is asymptomatic in humans. The results thereby provide insight and confidence in exon-skipped designs.

Myosin VI is an unconventional actin-based motor protein that challenges the classical lever arm hypothesis with its minus-end directed processive movement and unusually large step size, in spite of having a short lever arm with a single IQ domain. The presence of a unique insert (Insert 2) and rearrangement in the converter subdomain can account for its reverse directionality and large powerstroke. However, these adaptations are not sufficient in explaining the 30-36nm step sizes of myosin VI which is typically characteristic of motor proteins with longer lever arms, viz. myosin V with 6 IQ domains. Though it is possible that myosin VI can function either as a monomer or a dimer in cells, based on our studies on the functional properties of the protein, it is likely that a dimeric protein will be a more efficient actin anchor and a processive transporter. We have shown that dimerization of full-length myosin VI can be triggered by cargo binding and the cargo-bound motors walk processively on actin filaments with the expected step size. Our recent studies demonstrate that the region immediately distal to the lever arm of the myosin VI motor (the proximal tail) exists as a three-helix bundle and unfolds upon dimerization of two myosin VI monomers, therefore serving as a lever arm extension in myosin VI. Additionally, our studies show that the medial tail domain is necessary for dimerization since specific mutations in this region create constitutive monomers which are non-processive with no gating properties, indicating that dimerization is necessary for the proper functioning of myosin VI.

Myosin VI is one of 18 known classes of the molecular motor superfamily called myosin (1,2). All myosins rapidly bind and hydrolyze ATP in the presence or absence of actin. Until recently it was thought that all myosins moved toward the barbed (+) end of the actin filament. Myosin VI is the exception to that rule and may be unique among the myosin family members in that it moves toward the pointed (-) end of the actin filament (3). Our working model for myosin VI in a cell is that the full-length protein exists as a monomer if not bound to cargo. Binding of myosin VI monomers to cargo alters the conformation of the molecule, possibly exposing the high probability coiled-coil region (dimerization domain). Once dimerized, the myosin VI can move a vesicle processively toward the minus-end of an actin filament. GiPC and optineurin, two of the known myosin VI binding partners can dimerize, and thus potentially can initiate the dimerization of myosin VI when it binds. Both GiPC and optineurin has been expressed in insect Sf9 cells. Surface plasmon resonance (SPR) analysis showed that both GiPC and optineurin interact with full-length myosin VI within the nanomolar range. Both GiPC and optineurin when incubated with full-length myosin VI initiated its dimerization showed by ATPase assays, EM and TIRF microscopy. [1] Mermall V, Post PL, Mooseker MS. Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science. 279:527-33, 1998. [2] Sellers JR, Goodson HV: Motor proteins 2: myosins. Protein Profile 2:1323-1423, 1995. [3] Wells AL, Lin AW, Chen LQ, Safer D, Cain SM, Hasson T, Carragher BO, Milligan RA, Sweeney HL. Myosin VI is an actin-based motor that moves backwards. Nature. 401:505-8, 1999.

Myosin-II's rod-like tail drives filament assembly with a head arrangement that is often considered to be a symmetric bipole that generates equal and opposite contractile forces on actin. Self-assembled myosin filaments are shown here to be asymmetric in physiological buffer based on cross-correlated images from both atomic force microscopy and total internal reflection fluorescence. Quantitative cross-correlation of these orthogonal methods produces structural information unavailable to either method alone in showing that fluorescence intensity along the filament length is proportional to height. This implies that myosin heads form a shell around the filament axis, consistent with F-actin binding. A motor density of ∼50–100 heads/micrometer is further estimated but with an average of 32% more motors on one half of any given filament compared to the other, regardless of length. A purely entropic pyramidal lattice model is developed and mapped onto the Dyck paths problem that qualitatively captures this lack of length dependence and the distribution of filament asymmetries. Such strongly asymmetric bipoles are likely to produce an unbalanced contractile force in cells and in actin-myosin gels and thereby contribute to motility as well as cytoskeletal tension.

Myosin VI challenges the prevailing theory of how myosin motors move on actin: the lever arm hypothesis. While the reverse directionality and large powerstroke of myosin VI can be attributed to unusual properties of a subdomain of the motor (converter with a unique insert), these adaptations cannot account for the large step size on actin. Either the lever arm hypothesis needs modification, or myosin VI has some unique form of extension of its lever arm. We determined the structure of the region immediately distal to the lever arm of the motor and show that it is a three-helix bundle. Based on C-terminal truncations that display the normal range of step sizes on actin, CD, fluorescence studies, and a partial deletion of the bundle, we demonstrate that this bundle unfolds upon dimerization of two myosin VI monomers. This unconventional mechanism generates an extension of the lever arm of myosin VI.

Although myosin VI has properties that would allow it to function optimally as a dimer, full-length myosin VI exists as a monomer in isolation. Based on the ability of myosin VI monomers to dimerize when held in close proximity, we postulated that cargo binding normally regulates dimerization of myosin VI. We tested this hypothesis by expressing a known dimeric cargo adaptor protein of myosin VI, optineurin, and the myosin VI-binding segment from a monomeric cargo adaptor protein, Dab2. In the presence of these adaptor proteins, full-length myosin VI has ATPase properties of a dimer, appears as a dimer in electron micrographs, and moves processively on actin filaments. The results support a model in which cargo binding exposes internal dimerization sequences within full-length myosin VI. Because, unexpectedly, a monomeric fragment of Dab2 triggers dimerization, it would appear that myosin VI is designed to function as a dimer in cells.

Myosin VI is an unconventional motor protein that can move processively along the actin filament in an opposite direction towards the minus-end, contrary to all other known myosins. Despite its short lever arm, represented by a single IQ domain, myosin VI demonstrates large step sizes (30-36nm), typically characteristic of motor proteins with longer lever arms, viz. myosin V with 6 IQ domains. In cells, myosin VI is involved in diverse functions including Golgi transport, endocytosis and stereocilia maintenance. Though it is possible that myosin VI can function either as a dimer or a monomer in cells, based on our studies on the functional properties of the protein, it is likely that a dimeric protein can undergo intramolecular strain to become a more efficient actin anchor which makes it more competent as a transporter. Previous studies from our lab have shown that both full-length as well HMM fragments are capable of forming stable, processive dimers upon clustering, indicating that myosin VI monomers need to be in close proximity to initiate dimerization. Our recent studies show dimerization of full-length myosin VI can be triggered by cargo binding and the cargo-bound motors walk processively on actin filaments with the expected step size. Following the IQ motif, the lever arm extension of about hundred amino acid residues contains the sequence sufficient for dimerization. However, the accurate location of dimer formation remains controversial since the putative dimerization domain in myosin VI has non-native coiled-coil sequences. Our working hypothesis is that dimerization triggers the unfolding of a 3-helix bundle creating the 12nm extension required for proper myosin VI walking. Based on a series of truncations, we are in the process of testing this hypothesis and defining the nature and sequence of the dimerization domain.

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (

The β-thymosins are a highly conserved family of strongly polar ~5 kDa polypeptides that are widely distributed in metazoan cells (Fig. 1). Thymosin βin4, the most abundant and best-characterized β-thymosin, binds monomeric actin in a stable 1:1 complex and acts as an actin “buffer,” preventing spontaneous polymerization but supplying high concentrations of free actin monomers for rapid filament elongation when cells are stimulated by extracellular cues. Several biological regulatory effects are attributed to Tβin4 and oxidized Tβin4. Among these are the induction of angiogenesis, tumor metastasis and the inhibition of inflammation. Correspondingly, several therapeutic applications for Tβ4 have been proposed.