Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Size Dispersion of the Filler Particles and Its Consequences on the Light-Extinction Properties of TiO2/PET Nanocomposite Fibers.

Titanium dioxide (TiO2) nanoparticles have been extensively used to modify the optical properties of various types of materials. In particular, they have been intensively loaded onto polymer fibers to quench the light reflection. In situ polymerization and online addition are two common strategies for fabricating TiO2-loaded polymer nanocomposite fibers. The former does not require separate preparation of masterbatches as the latter does and therefore has its advantages in terms of decreasing the fabrication steps and economic costs. Moreover, it has been found that in situ-polymerized TiO2-loaded polymer nanocomposite fibers (e.g., TiO2/poly(ethylene terephthalate) fibers) usually have enhanced light-extinction properties over those prepared by the online addition process. Intuitively, there should be a difference in the filler particle dispersion for the two fabrication processes. This hypothesis has not yet been tackled due to the technical difficulty in acquiring the three-dimensional (3D) filler morphology inside the fiber matrix. In this paper, we report a study using the powerful focused ion beam-scanning electron microscopy (FIB-SEM) with a resolution of 20 nm to directly acquire the 3D microstructure of TiO2/poly(ethylene terephthalate) nanocomposite (TiO2/PET) fibers. This microscopy technique allows us to characterize the particle size statistics and the dispersion inside TiO2/PET fibers. We have found that the particle size of TiO2 inside the fiber matrix can be well modeled by Weibull statistics. Surprisingly, we find that TiO2 nanoparticles form more significant agglomeration in the in situ-polymerized TiO2/PET fibers. This observation is contrary to our common understanding of the two fabrication processes. Namely, slightly altering the particle dispersion with increased TiO2 filler size helps improve the light-extinction properties. The slightly increased filler size may have altered the Mie scattering between the nanoparticles and the incident visible light, leading to enhanced light-extinction properties of in situ-polymerized TiO2/PET nanocomposite fibers.

The inactivation domain of STIM1 acts through intramolecular binding to the coiled-coil domain in the resting state.

Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway that is controlled by the ER Ca2+ sensor STIM1. Abnormal activation of STIM1 directly influences Ca2+ influx, resulting in severe diseases such as Stormorken syndrome. The inactivation domain of STIM1 (IDstim) has been identified as being essential for Ca2+-dependent inactivation of STIM1 (CDI) after SOCE occurs. However, it is unknown whether IDstim is involved in keeping STIM1 inactive before CDI. Herein, we show that IDstim helps STIM1 keep inactive through intramolecular binding with the coiled-coil domain. Between IDstim and the coiled-coil domain, we found a short conserved linker whose extension or mutation leads to the constitutive activation of STIM1. We have demonstrated that IDstim needs the coiled-coil domain 1 (CC1) to inhibit the Ca2+ release-activated Ca2+ (CRAC) activation domain (CAD) activity and binds to a CC1-CAD fragment. Serial deletion of CC1 revealed that CC1α1 is a co-inhibitory domain of IDstim. CC1α1 deletion or leucine mutation, which abolishes the closed conformation, impaired the inhibitory effect and binding of IDstim. These results suggest that IDstim cooperates with CC1α1 to help STIM1 keep inactive under resting conditions.

Simultaneous Real-Time Three-Dimensional Localization and FRET Measurement of Two Distinct Particles.

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.

Increased Confinement and Polydispersity of STIM1 and Orai1 after Ca2+ Store Depletion.

STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca2+ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca2+ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca2+ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca2+ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility-measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient-decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca2+ influx, which can facilitate the rapid activation of local Ca2+ signaling pathways and the efficient replenishing of Ca2+ store in the ER in store-operated Ca2+ entry.

Simultaneous tracking of two motor domains reveals near simultaneous steps and stutter steps of myosin 10 on actin filament bundles.

Myosin X (Myo10) has several unique design features including dimerization via an anti-parallel coiled coil and a long lever arm, which allow it to preferentially move on actin bundles. To understand the stepping behavior of single Myo10 on actin bundles, we labeled two heads of Myo10 dimers with different fluorophores. Unlike previously described for myosin V (Myo5) and VI (Myo6), which display alternating hand-over-hand stepping, Myo10 frequently took near simultaneous steps of both heads, and less frequently, 2-3 steps of one head before the other head stepped. We suggest that this behavior results from the unusual kinetic features of Myo10, in conjunction with the structural properties of the motor domain/lever arm, which will favor movement on actin bundles rather than on single filaments.

Real-time three-dimensional tracking of single synaptic vesicles reveals that synaptic vesicles undergoing kiss-and-run fusion remain close to their original fusion site before reuse.

The release of neurotransmitters via the fusion between synaptic vesicles and the presynaptic membrane is an essential step in synaptic transmission. Synaptic vesicles generally undergo two distinct modes of exocytosis called full-collapse fusion and kiss-and-run fusion. In kiss-and-run fusion, the fusion pore of the synaptic vesicle opens transiently without the vesicle collapsing fully into the plasma membrane; thus, each synaptic vesicle can be used multiple times to release neurotransmitters. Despite considerable research, the detailed mechanisms that underlie kiss-and-run fusion remain elusive, particularly the location of synaptic vesicles after kiss-and-run events. To address this question, we performed real-time three-dimensional tracking of single synaptic vesicles labeled with a single quantum dot in the presynaptic terminal of cultured hippocampal neurons and analyzed the three-dimensional trajectories of these vesicles undergoing kiss-and-run fusion. We found that the majority of these synaptic vesicles underwent another exocytosis event within 120 nm of their original fusion site and underwent a second exocytosis event within 10 s of the first fusion event. These results indicate that after kiss-and-run fusion, synaptic vesicles remain relatively close to their original fusion site and can release repeatedly at brief intervals, allowing neurons to maintain neurotransmitter release during bursting activity.

Real-time subpixel-accuracy tracking of single mitochondria in neurons reveals heterogeneous mitochondrial motion.

Mitochondria are essential for cellular survival and function. In neurons, mitochondria are transported to various subcellular regions as needed. Thus, defects in the axonal transport of mitochondria are related to the pathogenesis of neurodegenerative diseases, and the movement of mitochondria has been the subject of intense research. However, the inability to accurately track mitochondria with subpixel accuracy has hindered this research. Here, we report an automated method for tracking mitochondria based on the center of fluorescence. This tracking method, which is accurate to approximately one-tenth of a pixel, uses the centroid of an individual mitochondrion and provides information regarding the distance traveled between consecutive imaging frames, instantaneous speed, net distance traveled, and average speed. Importantly, this new tracking method enables researchers to observe both directed motion and undirected movement (i.e., in which the mitochondrion moves randomly within a small region, following a sub-diffusive motion). This method significantly improves our ability to analyze the movement of mitochondria and sheds light on the dynamic features of mitochondrial movement.

A numerical method for resolving finite mixtures of three-dimensional inter-site distance distributions measured by single-molecule localization microscopy.

The three-dimensional (3D) inter-site distance can be measured by single-molecule localization microscopy. Existing theories and analysis tools for 3D inter-site distance measurement only consider the simplest case where all measured distances are from an identical 3D Rician distribution. There are many problems where the 3D inter-site distance measurement result is made up of multiple components, for example, the measurement of intramolecular distances of deoxyribonucleic acid with multiple possible conformations. In these cases, the overall distance distributions become finite mixtures of 3D Rician distributions (or 3D Rician mixtures). Here, we provide a numerical method using the 3D Rician mixture model to resolve the finite 3D inter-site distance mixtures, which is based on the expectation-maximization algorithm. The proposed method has been tested on simulation data of finite 3D inter-site distance mixtures. The result using the Gaussian mixture model in the developed method is also discussed for comparison.

The Antiparallel Coiled-Coil Domain Allows Multiple Forward Step Sizes of Myosin X.

Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of ∼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.

Decreased BDNF Release in Cortical Neurons of a Knock-in Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an increase in CAG repeats in the Huntingtin gene (HTT). The striatum is one of the most vulnerable brain regions in HD, and altered delivery of BDNF to the striatum is believed to underlie this high vulnerability. However, the delivery of BDNF to the striatum in HD remains poorly understood. Here, we used real-time imaging to visualize release of BDNF from cortical neurons cultured alone or co-cultured with striatal neurons. BDNF release was significantly decreased in the cortical neurons of zQ175 mice (a knock-in model of HD), and total internal reflection fluorescence microscopy revealed several release patterns of single BDNF-containing vesicles, with distinct kinetics and prevalence, in co-cultured cortical HD neurons. Notably, a smaller proportion of single BDNF-containing vesicles underwent full release in HD neurons than in wild-type neurons. This decreased release of BDNF in cortical neurons might lead to decreased BDNF levels in the striatum because the striatum receives BDNF mainly from the cortex. In addition, we observed a decrease in the total travel length and speed of BDNF-containing vesicles in HD neurons, indicating altered transport of these vesicles in HD. Our findings suggest a potential mechanism for the vulnerability of striatal neurons in HD and offer new insights into the pathogenic mechanisms underlying the degeneration of neurons in HD.

Altered Synaptic Vesicle Release and Ca2+ Influx at Single Presynaptic Terminals of Cortical Neurons in a Knock-in Mouse Model of Huntington's Disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the abnormal expansion of CAG repeats in the huntingtin (HTT) gene, which leads to progressive loss of neurons starting in the striatum and cortex. One possible mechanism for this selective loss of neurons in the early stage of HD is altered neurotransmission at synapses. Despite the recent finding that presynaptic terminals play an important role in HD, neurotransmitter release at synapses in HD remains poorly understood. Here, we measured synaptic vesicle release in real time at single presynaptic terminals during electrical field stimulation. We found the increase in synaptic vesicle release at presynaptic terminals in primary cortical neurons in a knock-in mouse model of HD (zQ175). We also found the increase in Ca2+ influx at presynaptic terminals in HD neurons during the electrical stimulation. Consistent with increased Ca2+-dependent neurotransmission in HD neurons, the increase in vesicle release and Ca2+ influx was rescued with Ca2+ chelators or by blocking N-type voltage-gated Ca2+ channels, suggesting N-type voltage-gated Ca2+ channels play an important role in HD. Taken together, our results suggest that the increased synaptic vesicles release due to increased Ca2+ influx at presynaptic terminals in cortical neurons contributes to the selective neurodegeneration of these neurons in early HD and provide a possible therapeutic target.

[Evaluation of methods for total nasal reconstruction].

OBJECTIVE: To evaluate the advantages, disadvantages and their indications of total nasal reconstruction with different techniques. METHODS: A series of total nasal reconstruction were treated with four methods from 1975 to 2003. These methods were tubed flap of arm,midline forehead flap with skin graft, midline forehead flap with bilateral frontotemporal flaps for repairing the donor site, and expanded forehead flap. RESULTS: All of the patients were treated successfully. The shape and function of the reconstructed noses were satisfactory. However, the traditional forehead flap with skin graft may leave a unsightly big and black scar on the forehead. The technique of the tubed flap of arm could provide enough tissue without remaining forehead scar and be easily shaped, but it required long period, multiple procedures and body fixation for three weeks. CONCLUSIONS: Midline forehead flap with bilateral frontotemporal flaps for repairing the donor site may be good for small nose reconstruction while expanded forehead flap could reconstruct a big nose. Tubed flap of arm may be used to the patients who do not wish to leave any scar on the forehead. Forehead flap with skin graft to repair the donor sit- should generally be avoided for nose reconstruction.