Kinetic analysis methods applied to single motor protein trajectories.

Molecular motors convert chemical or electrical energy into mechanical displacement, either linear or rotary. Under ideal circumstances, single-molecule measurements can spatially and temporally resolve individual steps of the motor, revealing important properties of the underlying mechanochemical process. Unfortunately, steps are often hard to resolve, as they are masked by thermal noise. In such cases, details of the mechanochemistry can nonetheless be recovered by analyzing the fluctuations in the recorded traces. Here, we expand upon existing statistical analysis methods, providing two new avenues to extract the motor step size, the effective number of rate-limiting chemical states per translocation step, and the compliance of the link between the motor and the probe particle. We first demonstrate the power and limitations of these methods using simulated molecular motor trajectories, and we then apply these methods to experimental data of kinesin, the bacterial flagellar motor, and F1-ATPase.

X-radiation enhances the collagen type I strap formation and migration potentials of colon cancer cells.

Rectal cancer treatment still fails with local and distant relapses of the disease. It is hypothesized that radiotherapy could stimulate cancer cell dissemination and metastasis. In this study, we evaluated the effect of X-radiation on collagen type I strap formation potential, i.e. matrix remodeling associated with mesenchymal cell migration, and behaviors of SW480, SW620, HCT116 p53+/+ and HCT116 p53-/- colon cancer cells. We determined a radiation-induced increase in collagen type I strap formation and migration potentials of SW480 and HCT116 p53+/+. Further studies with HCT116 p53+/+, indicated that after X-radiation strap forming cells have an increased motility. More, we detected a decrease in adhesion potential and mature integrin ß1 expression, but no change in non-muscle myosin II expression for HCT116 p53+/+ after X-radiation. Integrin ß1 neutralization resulted in a decreased cell adhesion and collagen type I strap formation in both sham and X-radiated conditions. Our study indicates collagen type I strap formation as a potential mechanism of colon cancer cells with increased migration potential after X-radiation, and suggests that other molecules than integrin ß1 and non-muscle myosin II are responsible for the radiation-induced collagen type I strap formation potential of colon cancer cells. This work encourages further molecular investigation of radiation-induced migration to improve rectal cancer treatment outcome.

Physiological and Histological Evaluations of the Cochlea between 3xTg-AD Mouse Model of Alzheimer's Diseases and R6/2 Mouse Model of Huntington's Diseases.

Patients with Alzheimer's diseases (AD) and Huntington's diseases (HD) are known to have abnormal auditory processing, but the physiological and histological evaluations of the cochlea between AD and HD have not been thoroughly assessed. Thus we assessed the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE), and then examined spiral ganglion neurons (SGNs) and hair cells in the cochlea using 3xTg-AD mouse model of AD and R6/2-HD mouse model of HD. We found that the threshold of ABR, but not DPOAE, was significantly increased in AD mice from 9 months of age and thereafter. The significant loss of SGNs, but not hair cells, was observed in the cochlea of 9- and 12-month AD mice. On the other hand, we found that both ABR and DPOAE thresholds were significantly increased in HD mice from 2 months of age and thereafter. The large loss of hair cells and the small loss of SGNs were observed in the cochlea of 3-month HD mice. Furthermore, the prestin expression in outer hair cells (OHCs) was significantly decreased in HD mice from 2 months of age and thereafter, and the loss of prestin expression was earlier before OHCs death in HD mice. Different from HD mice, the prestin expression in OHCs in AD mice was not changed even at 12 months of age. Our data suggest that cochlear pathology contributing to hearing loss is quite different between transgenic mice of AD and HD. More detailed pathological mechanisms for hearing loss between AD and HD need further study.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

High-speed atomic force microscopy: Structure and dynamics of single proteins.

For surface analysis of biological molecules, atomic force microscopy (AFM) is an appealing technique combining data acquisition under physiological conditions, for example buffer solution, room temperature and ambient pressure, and high resolution. However, a key feature of life, dynamics, could not be assessed until recently because of the slowness of conventional AFM setups. Thus, for observing bio-molecular processes, the gain of image acquisition speed signifies a key progress. Here, we review the development and recent achievements using high-speed atomic force microscopy (HS-AFM). The HS-AFM is now the only technique to assess structure and dynamics of single molecules, revealing molecular motor action and diffusion dynamics. From this imaging data, watching molecules at work, novel and direct insights could be gained concerning the structure, dynamics and function relationship at the single bio-molecule level.

Chemoreceptors and flagellar motors are subterminally located in close proximity at the two cell poles in spirochetes.

Green fluorescent protein (GFP) fusions, immunofluorescence microscopy, and cryo-electron tomography revealed that the chemoreceptors of the Lyme disease spirochete Borrelia burgdorferi form long, thin arrays near both cell poles. These arrays are in close proximity to the flagellar motors. This information provides a basis for further understanding motility, chemotaxis, and protein localization in spirochetes.

A mixture model for quantum dot images of kinesin motor assays.

We introduce a nearly automatic procedure to locate and count the quantum dots in images of kinesin motor assays. Our procedure employs an approximate likelihood estimator based on a two-component mixture model for the image data; the first component has a normal distribution, and the other component is distributed as a normal random variable plus an exponential random variable. The normal component has an unknown variance, which we model as a function of the mean. We use B-splines to estimate the variance function during a training run on a suitable image, and the estimate is used to process subsequent images. Parameter estimates are generated for each image along with estimates of standard errors, and the number of dots in the image is determined using an information criterion and likelihood ratio tests. Realistic simulations show that our procedure is robust and that it leads to accurate estimates, both of parameters and of standard errors.

Imaging single molecular motor motility with total internal reflection fluorescence microscopy (TIRFM).

Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules on or near the plasma membrane to be visualized with a very high signal-to-noise ratio. This strategy has been very successful for imaging molecular machines as they move and do work. We provide here a general protocol for imaging single molecular motors as they move along microtubule tracks. Our protocol is designed for the study of cytoplasmic dynein purified from Saccharomyces cerevisiae, but it represents a general framework for any in vitro single-molecule assay.

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a DeltamglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA-YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator.

Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins.

Yeast two-hybrid screens often produce vastly non-overlapping interaction data when the screens are conducted in different laboratories, or use different vectors, strains, or reporter genes. Here we investigate the underlying reasons for such inconsistencies and compare the effect of seven different vectors and their yeast two-hybrid interactions. Genome-wide array screens with 49 motility-related baits from Treponema pallidum yielded 77 and 165 interactions with bait vectors pLP-GBKT7 and pAS1-LP, respectively, including 21 overlapping interactions. In addition, 90 motility-related proteins from Escherichia coli were tested in all pairwise combinations and yielded 140 interactions when tested with pGBKT7g/pGADT7g vectors but only 47 when tested with pDEST32/pDEST22. We discuss the factors that determine these effects, including copy number, the nature of the fusion protein, and species-specific differences that explain non-conserved interactions among species. The pDEST22/pDEST32 vectors produce a higher fraction of interactions that are conserved and that are biologically relevant when compared with the pGBKT7/pGADT7-related vectors, but the latter appear to be more sensitive and thus detect more interactions overall.

Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins.

Type IV pili (T4P) are surface structures that undergo extension/retraction oscillations to generate cell motility. In Myxococcus xanthus, T4P are unipolarly localized and undergo pole-to-pole oscillations synchronously with cellular reversals. We investigated the mechanisms underlying these oscillations. We show that several T4P proteins localize symmetrically in clusters at both cell poles between reversals, and these clusters remain stationary during reversals. Conversely, the PilB and PilT motor ATPases that energize extension and retraction, respectively, localize to opposite poles with PilB predominantly at the piliated and PilT predominantly at the non-piliated pole, and these proteins oscillate between the poles during reversals. Therefore, T4P pole-to-pole oscillations involve the disassembly of T4P machinery at one pole and reassembly of this machinery at the opposite pole. Fluorescence recovery after photobleaching experiments showed rapid turnover of YFP-PilT in the polar clusters between reversals. Moreover, PilT displays bursts of accumulation at the piliated pole between reversals. These observations suggest that the spatial separation of PilB and PilT in combination with the noisy PilT accumulation at the piliated pole allow the temporal separation of extension and retraction. This is the first demonstration that the function of a molecular machine depends on disassembly and reassembly of its individual parts.

DNA molecule manipulation by motor proteins for analysis at the single-molecule level.

Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the lambdaDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin-biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale.

Mass spectrometric analysis of microtubule co-sedimented proteins from rat brain.

Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures.

Historical hematology: May-Hegglin anomaly.

May-Hegglin anomaly is a rare autosomal dominant platelet disorder characterized by thrombocytopenia, giant platelets, and unique leukocyte inclusion bodies. This disorder was first described by May, a German physician, in 1909, and was subsequently described by a Swiss physician, Hegglin, in 1945. The pathogenesis of the disorder had been unknown until recently, when mutations in the gene encoding for nonmuscle myosin heavy chain IIA (MYH9) were identified. Unique cytoplasmic inclusion bodies are aggregates of nonmuscle myosin heavy chain IIA, and are only present in granulocytes. It is not yet known why inclusion bodies are not present in platelets, monocytes, and lymphocytes, or how giant platelets are formed. Interestingly, MYH9 is also found to be responsible for several related disorders with macrothrombocytopenia and leukocytes inclusion, including Sebastian, Fechtner, and Epstein syndromes, which feature deafness, nephritis, and/or cataract. Current interest is centered upon the mechanisms by which a single mutation causes a variety of phenotypes.

NXF2 is involved in cytoplasmic mRNA dynamics through interactions with motor proteins.

Tap/NXF1, the founding member of the evolutionarily conserved NXF (Nuclear RNA export Factor) family of proteins, is required for the nuclear export of bulk poly(A)+ RNAs. In mice, three additional NXF family genes (NXF2, NXF3, NXF7) have been identified and characterized to date. Cumulative data suggest that NXF family members play roles, not only in nuclear mRNA export, but also in various aspects of post-transcriptional mRNA metabolism. In order to better understand the functional role of NXF2, we searched for its binding partners by yeast two-hybrid screening and identified several cytoplasmic motor proteins, including KIF17. The interaction of NXF2 with KIF17, which was confirmed by GST pull-down and co-immunoprecipitation assays, is mediated by the N-terminal domain of NXF2, which is required for the punctate localization patterns in dendrites of primary neurons. We also found that the NXF2-containing dendritic granules, which were co-localized with KIF17, mRNA and Staufen1, a known component of neuronal mRNA granules, moved bidirectionally along dendrites in a microtubule-dependent manner. These results suggest that NXF2, a nucleo-cytoplasmic mRNA transporter, plays additional roles in the cytoplasmic localization of mRNAs through interactions with cytoplasmic motor proteins.

Evidence that focal adhesion complexes power bacterial gliding motility.

The bacterium Myxococcus xanthus has two motility systems: S motility, which is powered by type IV pilus retraction, and A motility, which is powered by unknown mechanism(s). We found that A motility involved transient adhesion complexes that remained at fixed positions relative to the substratum as cells moved forward. Complexes assembled at leading cell poles and dispersed at the rear of the cells. When cells reversed direction, the A-motility clusters relocalized to the new leading poles together with S-motility proteins. The Frz chemosensory system coordinated the two motility systems. The dynamics of protein cluster localization suggest that intracellular motors and force transmission by dynamic focal adhesions can power bacterial motility.

The Vibrio motor proteins, MotX and MotY, are associated with the basal body of Na-driven flagella and required for stator formation.

The four motor proteins PomA, PomB, MotX and MotY, which are believed to be stator proteins, are essential for motility by the Na(+)-driven flagella of Vibrio alginolyticus. When we purified the flagellar basal bodies, MotX and MotY were detected in the basal body, which is the supramolecular complex comprised of the rotor and the bushing, but PomA and PomB were not. By antibody labelling, MotX and MotY were detected around the LP ring. These results indicate that MotX and MotY associate with the basal body. The basal body had a new ring structure beneath the LP ring, which was named the T ring. This structure was changed or lost in the basal body from a DeltamotX or DeltamotY strain. The T ring probably comprises MotX and MotY. In the absence of MotX or MotY, we demonstrated that PomA and PomB were not localized to a cell pole. From the above results, we suggest that MotX and MotY of the T ring are involved in the incorporation and/or stabilization of the PomA/PomB complex in the motor.

The distribution of motor proteins in the muscles and flame cells of the Schistosoma mansoni miracidium and primary sporocyst.

Schistosoma mansoni eggs, miracidia and primary sporocysts were labelled with phalloidin-rhodamine to visualize filamentous actin structures. Analysis of these forms by confocal fluorescence microscopy revealed the presence of previously well-defined circular and longitudinal muscle layers. Besides these muscular layers that sustain and provide motility to these parasite forms, we found in these 3 consecutive developmental stages of the parasite previously unidentified actin-rich tubular structures. In the 3 forms, 4 actin-rich tubules could be observed by optical sectioning underneath the well-developed muscle layers. The tubules appear in pairs, transversal to the length of the parasite, and located towards the extremities. By using an anti-flame cell specific antibody we confirmed that the tubules co-localize with flame cells and also determined that the tubule core is filled with microtubules. The additional presence of myosin in these tubules strongly suggests that they are contractile structures.