Array Of Filaments Research Articles

Ultracompact Lens‐Less “Spectrometer in Fiber” Based on Chirped Filament‐Array Gratings

Femtosecond laser irradiation is applied to a single‐mode optical fiber to embed a filament array through the silica cladding and guiding core and form chirped Bragg gratings. Unlike a planar‐shaped refractive index modification, the long and uniform filament facilitates efficient optical scattering into azimuthally narrowed radiation modes, external and transverse to the fiber cladding. Chirping of the grating period further provides spectral focusing. The combined spectral and azimuthal focusing permits lens‐less recording of bright and high‐resolution spectra spanning across most of the visible band with a low‐cost charged coupled device camera. The flexible point‐by‐point writing enables fiber tapping of light with engineered spectral and geometric focusing properties, permitting the design of new compact photonic devices based on the all‐fiber spectrometer.

Microtubules provide guidance cues for myofibril and sarcomere assembly and growth.

Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. Our observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.

Effect of 3D printing raster angle on reversible thermo-responsive composites using PLA/paper bilayer

In this study, an efficient fabrication method to realize a reversible thermo-responsive composite (TRC) and the effect of raster angle were investigated. For the facile fabrication of reversible TRCs, polylactic acid and commercial printing paper were used to generate a polymer/paper bilayer. Using the fused deposition modeling method of three-dimensional printing, three types of TRCs were fabricated and compared depending on the printing angle condition. Above the glass transition temperature, the polymer is relaxed, allowing straightening by the constraining paper layer; subsequently, it recovers its initial shape during cooling. Because the deposited filament arrays shrank in the longitudinal direction and expanded in another direction during heating, the raster angle was considered for better actuating performance. Thus, horizontal-type TRCs exhibited a greater range of average actuating performance compared with vertical-type TRCs (up to ∼24.7% increase in changing rate). Hence, this study supports a rapid, low-cost, and efficient fabrication method for soft robotics applications, including smart devices, grippers, and actuators.

Coordination in Coupled Arrays of Stiff Filaments—Modelling and Simulation

We present the mechanical model of an array of elastic filaments and simulate the response with different mechanical couplings. This class of systems is inspired by robust and elegant solutions for locomotion mechanics that have emerged in several small-scale biological entities in the form of beating protrusions, such as cellular cilia and eukaryotic flagella. The collective dynamics of cilia arrays reveals important features such as array alignments, two-phase asymmetric beating of individual filaments, and the emergence of metachronal coordination, which make them suitable for bio-inspired terrestrial and aquatic locomotion. The model presented here is the basis for further developments towards the design of terrestrial and aquatic locomotion systems for general purpose robotic devices.

Actin Cell Cortex: Structure and Molecular Organization.

The actin cytoskeleton consists of structurally and biochemically different actin filament arrays. Among them, the actin cortex is thought to have key roles in cell mechanics, but remains a poorly characterized part of the actin cytoskeleton. The cell cortex is typically defined as a thin layer of actin meshwork that uniformly underlies the plasma membrane of the entire cell. However, this definition applies only to specific cases. In general, the cortex structure and subcellular distribution vary significantly across cell types and physiological states of the cell. In this review, I focus on our current knowledge of the structure and molecular composition of the cell cortex.

Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays.

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

Atypical Myosin Tunes Dendrite Arbor Subdivision

Dendrite arbor pattern determines the functional characteristics of a neuron. It is founded on primary branch structure, defined through cell intrinsic and transcription-factor-encoded mechanisms. Developing arbors have extensive acentrosomal microtubule dynamics, and here, we report an unexpected role for the atypical actin motor Myo6 in creating primary branch structure by specifying the position, polarity,and targeting of these events. We carried out invivo time-lapse imaging of Drosophila adult sensory neuron differentiation, integrating machine-learning-based quantification of arbor patterning with molecular-level tracking of cytoskeletal remodeling. This revealed that Myo6 and the transcription factor Knot regulate transient surges of microtubule polymerization at dendrite tips; they drive retrograde extension of an actin filament array that specifies anterograde microtubule polymerization and guides these microtubules to subdivide the tip into multiple branches. Primary branches delineate functional compartments; this tunable branching mechanism is key to define and diversify dendrite arbor compartmentalization.

The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1)

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

Turbulence generation through an iterative cascade of the elliptical instability.

The essence of turbulent flow is the conveyance of energy through the formation, interaction, and destruction of eddies over a wide range of spatial scales-from the largest scales where energy is injected down to the smallest scales where it is dissipated through viscosity. Currently, there is no mechanistic framework that captures how the interactions of vortices drive this cascade. We show that iterations of the elliptical instability, arising from the interactions between counter-rotating vortices, lead to the emergence of turbulence. We demonstrate how the nonlinear development of the elliptical instability generates an ordered array of antiparallel secondary filaments. The secondary filaments mutually interact, leading to the formation of even smaller tertiary filaments. In experiments and simulations, we observe two and three iterations of this cascade, respectively. Our observations indicate that the elliptical instability could be one of the fundamental mechanisms by which the turbulent cascade develops.

Mitochondrial dynamics and segregation during the asymmetric division of Arabidopsis zygotes

The zygote is the first cell of a multicellular organism. In most angiosperms, the zygote divides asymmetrically to produce an embryo-precursor apical cell and a supporting basal cell. Zygotic division should properly segregate symbiotic organelles, because they cannot be synthesized de novo. In this study, we revealed the real-time dynamics of the principle source of ATP biogenesis, mitochondria, in Arabidopsis thaliana zygotes using live-cell observations and image quantifications. In the zygote, the mitochondria formed the extended structure associated with the longitudinal array of actin filaments (F-actins) and were polarly distributed along the apical-basal axis. The mitochondria were then temporally fragmented during zygotic division, and the resulting apical cells inherited mitochondria at higher concentration compared to the basal cells. Further observation of postembryonic organs showed that these mitochondrial behaviours are characteristic of the zygote. Overall, our results showed that the zygote has spatiotemporal regulation that unequally distributes the mitochondria.

Hydrodynamic interactions of filaments polymerizing against obstacles.

Polymerization and depolymerization of cytoskeletal filaments against cellular structures can generate forces that are key to many cellular processes, such as cell motility and chromosomes movements during cell division. Motions generated by these forces induce global cytoplasmic flows and couple the dynamics of the polymerizing filaments and other bodies immersed in the fluid through their long-range hydrodynamic interactions (HIs). Previous theoretical and computational studies have largely ignored HIs. We use three dimensional discrete simulations to study the relationship between polymerization forces and their resulting flows and HIs. As a model system, we choose a filament that is polymerizing against an obstacle, and is embedded in a cylindrical array of parallel filaments of the same length. We consider three distinct mechanical scenarios for the filaments within the array: (a) all of the filaments are polymerizing with the same velocity; (b) they are all fixed in space, and (c) they are freely suspended. We show that each of these conditions produce their unique cytoplasmic flows and each result in differentiable polymerization forces and velocities. We also study the effect of buckling of filaments on polymerization forces and velocities and discuss the effect of HIs on the onset of buckling transition. Finally, we show that HIs result in the bundling of the buckled filaments within the array.

Thin Filament Pyrometry Field Measurements in a Medium-Scale Pool Fire

This paper presents the development of a thin filament pyrometry method to characterize the time-varying temperature field in a medium-scale pool fire burning in a quiescent environment. A digital camera with optical filters and zoom lens was used to record the high temperature emission intensity of 14 μm diameter, Silicon-Carbide filaments oriented horizontally at various heights above the center of a steadily burning 0.30 m diameter methyl alcohol (methanol; CH3OH) pool fire. Experiments collected 30 Hz video of the planar filament array. In a separate experiment, a 50 μm diameter thermocouple was used to acquire independent temperature measurements in the high temperature zone of the fire. A correlation was developed between the probability density functions of the radiation-corrected thermocouple measurements and the camera grayscale pixel intensity of the filaments. This arrangement enables measurement of the time-varying temperature field over a temperature range from about 1150 K to 1900 K with a spatial resolution of 160 μm, a temporal resolution of 0.033 s, and an expanded uncertainty of about 150 K (at a mean temperature of 1300 K). Measurements of the grayscale pixel intensities of the filaments were obtained. False color maps of the temperature field were produced to characterize the high temperature field as a function of time. Using statistical analysis, the local time-averaged temperatures and their variance for each location on the filaments were determined. Time-averaged temperatures were compared favorably to previously reported measurements. The dominant frequency of the puffing fire was determined. The temperature field time series was transformed to consider its character during consecutive phases of the fire's puffing cycle. The analysis emphasizes the cyclic nature of a pool fire, providing insight on its complex dynamic structure.

Ultrafast laser burst-train filamentation for non-contact scribing of optical glasses.

A systematic study of glass scribing is presented on the benefits of ultrafast laser burst trains in generating filamentation tracks to guide cleaving of glass substrates. The interplay of Kerr self-focusing, plasma defocusing, and burst-train accumulation effects in filament formation was characterized by time-resolved in-situ microscopic imaging. Various filament-track scribing geometries were compared with and without assistance from burst-train pulse delivery or surface V-groove ablation. The cleaving guidance and reproducibility were examined together with the breaking force, facet morphology and flexural strength of cleaved substrates to assess the overall scribing and cleaving quality. The reported results attest to the benefits and flexibility of burst-mode ultrafast laser interactions to assist cleaving of optically transparent materials along well formed filament arrays.

Cardiac microtubules in health and heart disease.

Cardiomyocytes are large (∼40,000 µm3), rod-shaped muscle cells that provide the working force behind each heartbeat. These highly structured cells are packed with dense cytoskeletal networks that can be divided into two groups—the contractile (i.e. sarcomeric) cytoskeleton that consists of filamentous actin-myosin arrays organized into myofibrils, and the non-sarcomeric cytoskeleton, which is composed of β- and γ-actin, microtubules, and intermediate filaments. Together, microtubules and intermediate filaments form a cross-linked scaffold, and these networks are responsible for the delivery of intracellular cargo, the transmission of mechanical signals, the shaping of membrane systems, and the organization of myofibrils and organelles. Microtubules are extensively altered as part of both adaptive and pathological cardiac remodeling, which has diverse ramifications for the structure and function of the cardiomyocyte. In heart failure, the proliferation and post-translational modification of the microtubule network is linked to a number of maladaptive processes, including the mechanical impediment of cardiomyocyte contraction and relaxation. This raises the possibility that reversing microtubule alterations could improve cardiac performance, yet therapeutic efforts will strongly benefit from a deeper understanding of basic microtubule biology in the heart. The aim of this review is to summarize the known physiological roles of the cardiomyocyte microtubule network, the consequences of its pathological remodeling, and to highlight the open and intriguing questions regarding cardiac microtubules.Impact statementAdvancements in cell biological and biophysical approaches and super-resolution imaging have greatly broadened our view of tubulin biology over the last decade. In the heart, microtubules and microtubule-based transport help to organize and maintain key structures within the cardiomyocyte, including the sarcomere, intercalated disc, protein clearance machinery and transverse-tubule and sarcoplasmic reticulum membranes. It has become increasingly clear that post translational regulation of microtubules is a key determinant of their sub-cellular functionality. Alterations in microtubule network density, stability, and post-translational modifications are hallmarks of pathological cardiac remodeling, and modified microtubules can directly impede cardiomyocyte contractile function in various forms of heart disease. This review summarizes the functional roles and multi-leveled regulation of the cardiac microtubule cytoskeleton and highlights how refined experimental techniques are shedding mechanistic clarity on the regionally specified roles of microtubules in cardiac physiology and pathophysiology.

Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle

Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish (Porichthys notatus), into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres.

Ultrastructure and dynamics of the actin-myosin II cytoskeleton during mitochondrial fission.

Mitochondrial fission involves preconstriction of the organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and nonmuscle myosin II through a mechanism that remains unclear, mostly, due to unknown cytoskeleton ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded into an interstitial cytoskeletal network containing abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise a part of this interstitial network. Nonmuscle myosin II is found adjacent to mitochondria without specific enrichments at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas myosin II clouds mostly fluctuate nearby. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure onto mitochondrial surface, thus initiating curvature-sensing mechanisms that complete mitochondrial constriction.

Polar vacuolar distribution is essential for accurate asymmetric division of Arabidopsis zygotes

In most flowering plants, the asymmetric cell division of the zygote is the initial step in establishing the apical-basal axis of the mature plant. The zygote is polarized, possessing the nucleus at the apical tip and large vacuoles at the basal end. Despite their known polar localization, whether the positioning of the vacuoles and the nucleus is coordinated and what the role of the vacuole is in the asymmetric zygotic division remain elusive. In the present study, we utilized a live-cell imaging system to visualize the dynamics of vacuoles during the entire process of zygote polarization in Arabidopsis Image analysis revealed that the vacuoles formed tubular strands around the apically migrating nucleus. They gradually accumulated at the basal region and filled the space, resulting in asymmetric distribution in the mature zygote. To assess the role of vacuoles in the zygote, we screened various vacuole mutants and identified that shoot gravitropism2 (sgr2), in which the vacuolar structural change was impaired, failed to form tubular vacuoles and to polarly distribute the vacuole. In sgr2, large vacuoles occupied the apical tip and thus nuclear migration was blocked, resulting in a more symmetric zygotic division. We further observed that tubular vacuole formation and asymmetric vacuolar distribution both depended on the longitudinal array of actin filaments. Overall, our results show that vacuolar dynamics is crucial not only for the polar distribution along actin filaments but also for adequate nuclear positioning, and consequently zygote-division asymmetry.

Dissecting the nucleotide binding properties of the septins from S. cerevisiae.

Septins are a conserved family of guanosine triphosphate (GTP)-binding proteins that assemble into an ordered array of filaments at the mother bud neck in Saccharomyces cerevisiae cells. They are present in all higher eukaryotes except plants. Septins belong structurally to the P-Loop nucleoside triphosphatase (NTPases) like Rab and Ras. However, unlike other small guanosine triphosphatase (GTPases) septins are supposed to act as scaffolds rather than signalling mediators. This is why they are considered as the fourth class of cytoskeletal proteins. It is assumed that septins fulfil their functions independently of the bound nucleotide. The role of guanosine diphosphosphate (GDP) and GTP binding and subsequent hydrolysis was controversial debated in the last couple of years. Lack of crystal structures of yeast septin subunits or rods and difficulties to isolate single monomeric septin subunits often hindered the correlation of results obtained from in vivo studies with biochemical data. Recently, nucleotide binding and hydrolysis was connected to the formation of septin rods from its subunits. However, the evidence was only indirectly obtained through the use of septin mutants in the context of intact cells. We provide here mechanistic insight into the nucleotide binding of the yeast septins by in vitro assays using purified septin rods and building blocks, thereby adding further insights to the already available models on septin filament formation.

Assembly of myosin II filament arrays: Network Contraction versus Expansion.

How cellular contractile systems assemble has fascinated scientists for generations. The major molecule responsible for cellular force generation is the molecular motor, non-muscle myosin II (NMII). NMII molecules are organized into single myosin filaments and larger arrays of filaments called NMII stacks, which are capable of generating increasing amounts of force. The textbook model of NMII stack assembly is the Network Contraction Model, where ensembles of distinct NMII filaments condense into a NMII stack by pulling on actin filaments. While this model has been widely accepted for ~20 years, it has been difficult to test inside cells due to the small size of NMII filaments. Recently, interest in how NMII stacks form has been reinvigorated by the advent of super-resolution microscopy techniques which have afforded unprecedented resolution of NMII filaments inside cells. A number of recent publications using these techniques have called into question key aspects of the Network Contraction Model, and our understanding of how NMII stacks assemble.

Structured illumination microscopy imaging reveals localization of replication protein A between chromosome lateral elements during mammalian meiosis

An important event enabling meiotic prophase I to proceed is the close juxtaposition of conjoined chromosome axes of homologs and their assembly via an array of transverse filaments and meiosis-specific axial elements into the synaptonemal complex (SC). During meiosis, recombination requires the establishment of a platform for recombinational interactions between the chromosome axes and their subsequent stabilization. This is essential for ensuring crossover recombination and proper segregation of homologous chromosomes. Thus, well-established SCs are essential for supporting these processes. The regulation of recombination intermediates on the chromosome axis/SC and dynamic positioning of double-strand breaks are not well understood. Here, using super-resolution microscopy (structured illumination microscopy), we determined the localization of the replication protein A (RPA) complex on the chromosome axes in the early phase of leptonema/zygonema and within the CEs of SC in the pachynema during meiotic prophase in mouse spermatocytes. RPA, which marks the intermediate steps of pairing and recombination, appears in large numbers and is positioned on the chromosome axes at the zygonema. In the pachynema, RPA foci are reduced but do not completely disappear; instead, they are placed between lateral elements. Our results reveal the precise structure of SC and localization dynamics of recombination intermediates on meiocyte chromosomes undergoing homolog pairing and meiotic recombination.