Cytoplasmic mechanical properties are often treated as constant background parameters, yet whether they change systematically during development remains unclear. Here, we directly measured cytoplasmic mechanics during early embryogenesis of Caenorhabditis elegans by establishing active microrheology using micrometer-sized magnetic droplets. Active microrheology revealed a progressive decrease in creep compliance from the 1-cell to the 8-cell stage, indicating a progressive stiffening of the local cytoplasmic environment during development. This decrease persisted even when cytokinesis was inhibited, demonstrating that it cannot be explained solely by geometric changes associated with cell division. Passive microrheology using 40-nm fluorescent beads showed a consistent decrease in probe mobility over development. Together, these results demonstrate that cytoplasmic mechanical properties undergo a gradual, developmentally programmed change during embryogenesis that cannot be explained by cell division-associated geometry alone.

Inside cells, molecular motors transport cargo through a highly crowded cytoplasmic environment. While such an environment is assumed to hinder transport, its precise effect remains unclear. Here, we investigated how the dynamics of cytoplasmic environments affect dynein-driven transport in C. elegans early embryos. In living embryos, we found that an artificial dynein-cargo complex exhibited significantly faster transport than in vitro, indicating an active acceleration mechanism in vivo. By altering the activity of actomyosin networks, we found that dynein-driven transport was accelerated by actomyosin-driven cytoplasmic fluctuations, with speed increasing upon myosin upregulation and decreasing upon its depletion. Furthermore, in vitro force measurements of dynein suggest that the asymmetric force response to random forces, generated by fluctuating dynamics of actomyosin networks, may contribute to acceleration. This study provides insights into a regulatory mechanism of molecular motors within fluctuating cytoplasm, harnessing cytoplasmic fluctuations to enhance transport efficiency in a highly crowded environment.

Cytoplasmic streaming is driven by molecular motors that move along the cytoskeleton and entrain the surrounding fluid. In certain cell types, the direction of cytoplasmic streaming is not predefined but rather stochastic. For example, in meiotic cytoplasmic streaming of zygotes, the direction of the swirl reverses over time [Kimura , ]. While a mean-field theory explained the reversal, the structure of flows in three-dimensional space and the mechanical role of the endoplasmic reticulum (ER) remains unknown. To test the hypothesis that the elastic ER bonds and hydrodynamic interactions between microtubules mediate the orientational order of microtubules, leading to directional cytoplasmic streaming, we computationally investigated fluid-structure interactions in cytoplasmic flow, ER networks, and microtubule structures in the presence of microtubule dynamic instability. The results indicate that the occasionally reversing swirls emerge within a certain range of ER elasticity, and our experimental measurements of the ER elasticity agree with the numerical predictions. Mass transport analysis further demonstrates that the reversing swirls are suitable for intracellular particle mixing. These findings illustrate the delicate balance of meiotic cytoplasmic streaming and provide insights into the physiology of the embryogenesis. Published by the American Physical Society 2025

Inside cells, molecular motors transport cargoes within the actively fluctuating environment known as the cytoplasm. How fluctuations in the cytoplasm affect motor-driven transport is not fully understood. In this study, we investigated the role of fluctuations for transport along microtubules using C. elegans early embryos, focusing on transport driven by cytoplasmic dynein. An artificial motor-cargo complex showed faster transport in vivo than in vitro , suggesting an in vivo acceleration mechanism. We also found that endogenous early endosome transport by dynein is significantly enhanced by the fluctuations in the cytoplasm, which is attributed to the activity of actomyosin networks. An in vitro force measurement of dynein suggests that the asymmetric force response would play a key role in the acceleration. This study provides insights into a regulatory mechanism of molecular motors within actively fluctuating cytoplasm, potentially utilizing random force originating from fluctuating dynamics in the cytoplasm to increase transport efficiency.### Competing Interest StatementThe authors have declared no competing interest.

Mechanical stress significantly affects the physiological functions of cells, including tissue homeostasis, cytoskeletal alterations, and intracellular transport. As a major cytoskeletal component, microtubules respond to mechanical stimulation by altering their alignment and polymerization dynamics. Previously, we reported that microtubules may modulate cargo transport by one of the microtubule-associated motor proteins, dynein, under compressive mechanical stress. Despite the critical role of tensile stress in many biological functions, how tensile stress on microtubules regulates cargo transport is yet to be unveiled. The present study demonstrates that the low-level tensile stress-induced microtubule deformation facilitates dynein-driven transport. We validate our experimental findings using all-atom molecular dynamics simulation. Our study may provide important implications for developing new therapies for diseases that involve impaired intracellular transport.

By facilitating a water/water phase separation (w/wPS), crowded biopolymers in cells form droplets that contribute to the spatial localization of biological components and their biochemical reactions. However, their influence on mechanical processes driven by protein motors has not been well studied. Here, we show that the w/wPS droplet spontaneously entraps kinesins as well as microtubules (MTs) and generates a micrometre-scale vortex flow inside the droplet. Active droplets with a size of 10–100 µm are generated through w/wPS of dextran and polyethylene glycol mixed with MTs, molecular-engineered chimeric four-headed kinesins and ATP after mechanical mixing. MTs and kinesin rapidly created contractile network accumulated at the interface of the droplet and gradually generated vortical flow, which can drive translational motion of a droplet. Our work reveals that the interface of w/wPS contributes not only to chemical processes but also produces mechanical motion by assembling species of protein motors in a functioning manner.

Cytoplasmic dynein is responsible for various cellular processes during the cell cycle. The mechanism by which its activity is regulated spatially and temporarily inside the cell remains elusive. There are various regulatory proteins of dynein, including dynactin, NDEL1/NUD-2, and LIS1. Characterizing the spatiotemporal localization of regulatory proteins in vivo will aid understanding of the cellular regulation of dynein. Here, we focused on spindle formation in the Caenorhabditis elegans early embryo, wherein dynein and its regulatory proteins translocated from the cytoplasm to the spindle region upon nuclear envelope breakdown (NEBD). We found that (i) a limited set of dynein regulatory proteins accumulated in the spindle region, (ii) the spatial localization patterns were distinct among the regulators, and (iii) the regulatory proteins did not accumulate in the spindle region simultaneously but sequentially. Furthermore, the accumulation of NUD-2 was unique among the regulators. NUD-2 started to accumulate before NEBD (pre-NEBD accumulation), and exhibited the highest enrichment compared to the cytoplasmic concentration. Using a protein injection approach, we revealed that the C-terminal helix of NUD-2 was responsible for pre-NEBD accumulation. These findings suggest a fine temporal control of the subcellular localization of regulatory proteins.

Mechanical stress on cells has profound influences on biological processes, such as cell shape regulation, the formation of tissue patterns, and development. Recently, mechanosensing properties of the microtubule, an important cytoskeletal component, have drawn attention. In this work, we studied cargo transport by dynein, a microtubule-associated motor protein, along microtubules deformed under mechanical stress. We reveal that the microtubule deformation took place as a response to the applied stress and that the deformation of microtubules facilitated the transport of dynein-driven quantum dots. This finding will provide opportunities to explore the role of microtubules as molecular mechanotransducers in cellular processes.

Cytoplasmic dynein-1 (hereafter referred to as dynein) is a major microtubule-based motor critical for cell division. Dynein is essential for the formation and positioning of the mitotic spindle as well as the transport of various cargos in the cell. A striking feature of dynein is that, despite having a wide variety of functions, the catalytic subunit is coded in a single gene. To perform various cellular activities, there seem to be different types of dynein that share a common catalytic subunit. In this review, we will refer to the different kinds of dynein as "dyneins." This review attempts to classify the mechanisms underlying the emergence of multiple dyneins into four layers. Inside a cell, multiple dyneins generated through the multi-layered regulations interact with each other to form a network of dyneins. These dynein networks may be responsible for the accurate regulation of cellular activities, including cell division. How these networks function inside a cell, with a focus on the early embryogenesis ofCaenorhabditis elegansembryos, is discussed, as well as future directions for the integration of our understanding of molecular layering to understand the totality of dynein's function in living cells.

Tubulin polymerization-promoting protein family member 3 (TPPP3) is a conserved protein factor found in cytoplasm associated with microtubules in many ciliated cells. The expression level of TPPP3 in mouse tracheal epithelial cells (mTEC) was reported to vary in concomitant with the formation of microtubule networks in the cytoplasm during epithelia development. These observations suggest that TPPP3 takes part in formation of the microtubule network. Here, we expressed several TPPP3 constructs and, by using conventional biochemical measurements and single molecule observations, examined its effects on cell morphism of HEK293 cells, the binding, the bundling, and the elongation of microtubules in vitro. TPPP3 formed microtubule networks in vitro in a concentration dependent manner and showed specific but weak binding (Kd = ca. 5 μM) to the microtubules. The molecular dissection combined with single molecule observations suggests that the ability to dimerize the TPPP3 molecules resides in the N-terminal domain and the ability to promote microtubule elongation resides in the C-terminal domain of TPPP3. Micro-rheology measurements of TPPP3-microtubule network were carried out, in which the Brownian motion of microspheres of 0.2 μm-diameter in the microtubule network was measured. The measurement showed that the network was crosslinked but remained flexible. This crosslinked flexibility enables the network to adopt the optimal mechanical configuration in response to the external force. These results suggest that during the development of the basal body array in ciliated cells, the loose but somehow restricted network provides the adaptability of the array to the external load. This work was supported by funding from the Dynamic Mechanisms and Fundamental Technology for Biological Systems and the Creation of Fundamental Technologies for Understanding and Control of Biosystem Dynamics, CREST, from Japan Science and Technology.

A cell can be best resembled by a metropolitan city where all its functions are driven by the interactions of densely packed proteins. For the traffic system in this ‘city’, the expressway consists of actin and microtubules (MTs). On the other hand, the stepping motor proteins serve as ‘vehicles’ and transport cargo from the nucleus (minus end) to the periphery (plus end) and vice versa [1]. Kinesin-1 and cytoplasmic dynein are the two major cytoplasmic motors that transport cargos towards the plus and minus end of the MT respectively. Stimulated or spontaneous cell contraction, or constrained MT polymerization at the cell periphery give rise to buckling deformation of MT in cells, that may result into buckling with short wavelengths (e.g., several µm) [2-5]. While significant advancement is made in understanding the role of kinesin in transporting cargo individually or as team along undeformed MT [6-8], how the deformity in the MT due to buckling may affect the motility of the motor is overlooked. Kinesin takes regular steps of 8 nm in a hand-over-hand fashion [9] whereas dynein shows variable step sizes ranging from 8 – 32 nm uncoordinatedly [10]. Advanced studies on the structure and mechanism still do not provide clear idea on how the uncoordinated stepping mechanism in dynein responds to the buckled MT. Therefore, the effect of buckling of MT on the cargo transportation has a large scope to explore. Previously, we developed an experimental set-up, micro-stretcher, to control buckling of the MT filaments on a 2D elastic medium [11]. Present study exploits the knowledge from the system of MT buckling to further explore its contribution on the dynein based cargo transportation in vitro. For this, we fixed MTs on a pre-stretched elastic substrate, polydimethylsiloxane (PDMS), through interaction with kinesins. Relaxation of the pre-stretched PDMS consequently developed axial compression stress on the MTs, resulting into their buckling. We controlled the release of tensile strain of the PDMS to tune the buckling extent of MTs. Next, we immobilized the MTs with mild glutaraldehyde treatment and allowed the transportation of Qdots by motors along the undeformed and deformed MTs. We analyzed different parameters, e.g. velocities, pauses and run lengths from the observation of the cargo transportation. With increasing MT buckling we observed changes in the cargo transportation behavior of the motors. For kinesins, we observed that, the cargo velocities decreased with increasing strain in the MTs and finally, cargo transportation was halted at high strain region. While for dynein, cargo transportation prevailed with increased overall velocity with two peaks, one occurring in the low and the other at high strain region. Our goal is to explore the mechanism of how the buckling deformation of MT is affecting the observed phenomena in motor protein-based cargo transportation. Structural deformation in MTs, particularly, have showed to implicate several neurodegenerative diseases such as Alzheimer's disease, cardiovascular syndromes, neurological disorders and cancers etc. [12]. Therefore in vitro study of motor proteins in transporting cargo along deformed MTs will contribute not only in understanding the mechanism of motor motility in cells but also to target specific pathways for various neurodegenerative diseases. References: Alberts B., et al., Molecular Biology of the Cell, 4th edition, Garland Science, Taylor & Francis Group, (2002) Odde D. J., et al., J Cell Sci 112, 3283–3288 (1999) Pampaloni, F., et al, PNAS, 103, 10248–10253 (2006) Heidemann, S. R., Kaech, S., Buxbaum, R. E., Matus, A., J Cell Biol, 145, 109–122, (1999) Wang, N. et al., PNAS, 98, 7765–7770, (2001) Brangwynne et al., J Cell Biol, 173, 733–741, (2006) Berger, F. et al., Biochem Soc Trans, 39, 1211–5, (2011) Beeg, J., et al., Biophys J, 94, 532–41 (2008) Gross, S. P., Vershinin, M., Shubeita, G. T., Curr Biol, 17, R478–86, (2007) Burgess S. A, et al., Nature, 421, 715–718 (2003) Kabir, A. M. R., Inoue, D., Afrin, T., Mayama. H., Sada. K. and Kakugo, A., Sci. Rep. 5:17222, DOI: 10.1038/srep17222 (2015). Cross A.R., Williams R.C. Jr., Cell Motil Cytoskeleton, 20, 272-278 (1991)

Cytoskeletal organization is essential for the precise morphogenesis of cells, tissues, and organs. Cytoskeletons, bound to scaffolding proteins, regulate the apical junction complex (AJC), which is composed of tight and adherens junctions, and located at the apical side of epithelial cell sheets. Cingulin is a tight junction-associated protein that binds to both actin filaments and microtubules. However, how cingulin binds to microtubules and whether cingulin can bind to actin and microtubules simultaneously are unclear. Here we examined the mechanisms behind cingulin’s cytoskeleton-binding properties. First, using total internal reflection fluorescence microscopy, we detected cingulin at microtubule cross points. We then found the interdomain interactions in cingulin molecules. Notably, we found that this interaction was regulated by AMPK-dependent phosphorylation and changed cingulin’s conformation and binding properties to actin filaments and microtubules. Finally, we found that the AMPK-regulated cingulin properties regulated the barrier functions of epithelial cell sheets. We propose that the cellular metabolic state, which involves AMPK, can contribute to the organization and maintenance of epithelial tissues through cingulin’s tight junction/cytoskeleton regulation.

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Microtubule (MT) networks play key roles in cell division, intracellular transport, and cell motility. These functions of MT networks occur through interactions between MTs and various associated proteins, notably motor proteins that bundle and slide MTs. Our objective in this study was to address the question of how motors determine the nature of MT networks. We conducted in vitro assays using homotetrameric kinesin Eg5, a motor protein involved in the formation and maintenance of the mitotic spindle. The mixing of Eg5 and MTs produced a range of spatiotemporal dynamics depending on the motor/filament ratio. Low motor/filament ratios produced globally connected static MT networks with sparsely distributed contractile active nodes (motor-accumulating points with radially extending MTs). Increasing the motor/filament ratio facilitated the linking of contractile active nodes and led to a global contraction of the network. When the motor/filament ratio was further increased, densely distributed active nodes formed local clusters and segmented the network into pieces with their strong contractile forces. Altering the properties of the motor through the use of chimeric Eg5, which has kinesin-1 heads, resulted in the generation of many isolated asters. These results suggest that the spatial distribution of contractile active nodes determines the dynamics of MT-motor networks. We then developed a coarse-grained model of MT-motor networks and identified two essential features for reproducing the experimentally observed patterns: an accumulation of motors that form the active nodes necessary to generate contractile forces, and a nonlinear dependency of contractile force on motor densities. Our model also enabled us to characterize the mechanical properties of the contractile network. Our study provides insight into how local motor-MT interactions generate the spatiotemporal dynamics of macroscopic network structures.

Microtubules (MTs) in cells form hierarchical network structure and play important roles in various cellular activities. They emerge as the result of the interactions between MTs and various types of MT-binding proteins, and dynamically changes their structures during cell cycles and according to physiological roles of the cell. To obtain an integrative perspective of the MT networks, we performed reconstruction of the MT networks by using MTs and bipolar kinesin, Eg5. Human Eg5 expressed by HEK293 cells moved processively on an MT at ca. 10 nm/s and had ability to bundle MTs in parallel and anti-parallel manners. When MTs and Eg5 were mixed above the critical concentrations of 1 μM and 1 nM, respectively, the mixture generated various types of network architecture depending on the kinesin-MT mixing ratios. At the low ratios (<1:350), the system generates static network staying in the same architecture for >20-hours. Increase to 1:250 made the network contractile with two distinct phases: early slow contraction followed by the abrupt shortening and rupture of the network. Further increase to 1:40, MT networks rapidly contracted into large aggregate immediately after addition of ATP. In contrast, modulation of motor property by using recombinant Eg5 with four heads replaced by faster kinesin (KIF5B, 140 nm/s) heads led to generate a large number of asters. A coarse-grained mathematical model reproduced the network dynamics experimentally observed and delivered a comprehensive picture on the range and diversity of Eg5-MT networks at different spatiotemporal scales. We also calculated the elastic energy stored in the networks, and found that it is accumulated through early contractions in the active network, and can be released into mechanical work during rupturing. This feature might play some roles in the initiation of cell locomotion. (Supported by CREST, JST).

Cytoplasmic dynein is a minus-end-directed microtubule-based motor involving various cellular functions including intracellular transport, cell division, and neuronal migration. Recent studies have revealed that single molecules of cytoplasmic dynein do not display unidirectional movement as observed in intracellular transport, but diffuse along microtubules. This diffusive behavior is transitioned to the unidirectional movement via regulatory protein mediated mechanism or self-regulated mechanism. Here, we review the recent advances in the study of cytoplasmic dynein regulation.

Furuta and colleagues report that single dynein molecules are kept in an autoinhibited state through the stacking of their two head domains. This autoinhibition is relieved when dynein molecules assemble together on cargo.