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1. Simulation of microtubule–cytoplasm interaction revealed the importance of fluid dynamics in determining the organization of microtubules

   https://doi.org/10.1002/pld3.505  

   Murshed, Mohammad ; Wei, Donghui ; Gu, Ying ; Wang, Jin ( July 2023 , Plant Direct)

   Although microtubules in plant cells have been extensively studied, the mechanisms that regulate the spatial organization of microtubules are poorly understood. We hypothesize that the interaction between microtubules and cytoplasmic flow plays an important role in the assembly and orientation of microtubules. To test this hypothesis, we developed a new computational modeling framework for microtubules based on theory and methods from the fluid–structure interaction. We employed the immersed boundary method to track the movement of microtubules in cytoplasmic flow. We also incorporated details of the encounter dynamics when two microtubules collide with each other. We verified our computational model through several numerical tests before applying it to the simulation of the microtubule–cytoplasm interaction in a growing plant cell. Our computational investigation demonstrated that microtubules are primarily oriented in the direction orthogonal to the axis of cell elongation. We validated the simulation results through a comparison with the measurement from laboratory experiments. We found that our computational model, with further calibration, was capable of generating microtubule orientation patterns that were qualitatively and quantitatively consistent with the experimental results. The computational model proposed in this study can be naturally extended to many other cellular systems that involve the interaction between microstructures and the intracellular fluid.

    

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2. Probing stress-regulated ordering of the plant cortical microtubule array via a computational approach

   https://doi.org/10.1186/s12870-023-04252-5  

   Li, Jing ; Szymanski, Daniel B. ; Kim, Taeyoon ( December 2023 , BMC Plant Biology)

   Morphological properties of tissues and organs rely on cell growth. The growth of plant cells is determined by properties of a tough outer cell wall that deforms anisotropically in response to high turgor pressure. Cortical microtubules bias the mechanical anisotropy of a cell wall by affecting the trajectories of cellulose synthases in the wall that polymerize cellulose microfibrils. The microtubule cytoskeleton is often oriented in one direction at cellular length-scales to regulate growth direction, but the means by which cellular-scale microtubule patterns emerge has not been well understood. Correlations between the microtubule orientation and tensile forces in the cell wall have often been observed. However, the plausibility of stress as a determining factor for microtubule patterning has not been directly evaluated to date.

   Here, we simulated how different attributes of tensile forces in the cell wall can orient and pattern the microtubule array in the cortex. We implemented a discrete model with transient microtubule behaviors influenced by local mechanical stress in order to probe the mechanisms of stress-dependent patterning. Specifically, we varied the sensitivity of four types of dynamic behaviors observed on the plus end of microtubules – growth, shrinkage, catastrophe, and rescue – to local stress. Then, we evaluated the extent and rate of microtubule alignments in a two-dimensional computational domain that reflects the structural organization of the cortical array in plant cells.

   Our modeling approaches reproduced microtubule patterns observed in simple cell types and demonstrated that a spatial variation in the magnitude and anisotropy of stress can mediate mechanical feedback between the wall and of the cortical microtubule array.

    

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3. Laser ablation and fluid flows reveal the mechanism behind spindle and centrosome positioning

   Wu, H. ; Kabacaoğlu, G. ; Nazockdast, E. ; Chang, H-C ; Shelley, M. J. ; Needleman, D. J. ( January 2024 , Nature physics)

   Few techniques are available for studying the nature of forces that drive subcellular dynamics. Here we develop two complementary ones. The first is femtosecond stereotactic laser ablation, which rapidly creates complex cuts of subcellular structures and enables precise dissection of when, where and in what direction forces are generated. The second is an assessment of subcellular fluid flows by comparison of direct flow measurements using microinjected fluorescent nanodiamonds with large-scale fluid-structure simulations of different force transduction models. We apply these techniques to study spindle and centrosome positioning in early Caenorhabditis elegans embryos and to probe the contributions of microtubule pushing, cytoplasmic pulling and cortical pulling upon centrosomal microtubules. Based on our results, we construct a biophysical model to explain the dynamics of centrosomes. We demonstrate that cortical pulling forces provide a general explanation for many behaviours mediated by centrosomes, including pronuclear migration and centration, rotation, metaphase spindle positioning, asymmetric spindle elongation and spindle oscillations. This work establishes methodologies for disentangling the forces responsible for cell biological phenomena. 

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4. Modeling the effects of lattice defects on microtubule breaking and healing

   https://doi.org/10.1002/cm.21346  

   Jiang, Nan ; Bailey, Megan E. ; Burke, Jessica ; Ross, Jennifer L. ; Dima, Ruxandra I. ( January 2017 , Cytoskeleton)

   Microtubule reorganization often results from the loss of polymer induced through breakage or active destruction by energy‐using enzymes. Pre‐existing defects in the microtubule lattice likely lower structural integrity and aid filament destruction. Using large‐scale molecular simulations, we model diverse microtubule fragments under forces generated at specific positions to locally crush the filament. We show that lattices with 2% defects are crushed and severed by forces three times smaller than defect‐free ones. We validate our results with direct comparisons of microtubule kinking angles during severing. We find a high statistical correlation between the angle distributions from experiments and simulations indicating that they sample the same population of structures. Our simulations also indicate that the mechanical environment of the filament affects breaking: local mechanical support inhibits healing after severing, especially in the case of filaments with defects. These results recall reports of microtubule healing after flow‐induced bending and corroborate prior experimental studies that show severing is more likely at locations where microtubules crossover in networks. Our results shed new light on mechanisms underlying the ability of microtubules to be destroyed and healed in the cell, either by external forces or by severing enzymes wedging dimers apart. © 2016 Wiley Periodicals, Inc.

    

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5. Trichoplusia ni Kinesin-1 Associates with Autographa californica Multiple Nucleopolyhedrovirus Nucleocapsid Proteins and Is Required for Production of Budded Virus

   https://doi.org/10.1128/JVI.02912-15  

   Biswas, Siddhartha ; Blissard, Gary W. ; Theilmann, David A. ; McFadden, G. ( March 2016 , Journal of Virology)

   ABSTRACT The mechanism by which nucleocapsids of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) egress from the nucleus to the plasma membrane, leading to the formation of budded virus (BV), is not known. AC141 is a nucleocapsid-associated protein required for BV egress and has previously been shown to be associated with β-tubulin. In addition, AC141 and VP39 were previously shown by fluorescence resonance energy transfer by fluorescence lifetime imaging to interact directly with the Drosophila melanogaster kinesin-1 light chain (KLC) tetratricopeptide repeat (TPR) domain. These results suggested that microtubule transport systems may be involved in baculovirus nucleocapsid egress and BV formation. In this study, we investigated the role of lepidopteran microtubule transport using coimmunoprecipitation, colocalization, yeast two-hybrid, and small interfering RNA (siRNA) analyses. We show that nucleocapsid AC141 associates with the lepidopteran Trichoplusia ni KLC and kinesin-1 heavy chain (KHC) by coimmunoprecipitation and colocalization. Kinesin-1, AC141, and microtubules colocalized predominantly at the plasma membrane. In addition, the nucleocapsid proteins VP39, FP25, and BV/ODV-C42 were also coimmunoprecipitated with T. ni KLC. Direct analysis of the role of T. ni kinesin-1 by downregulation of KLC by siRNA resulted in a significant decrease in BV production. Nucleocapsids labeled with VP39 fused with three copies of the mCherry fluorescent protein also colocalized with microtubules. Yeast two-hybrid analysis showed no evidence of a direct interaction between kinesin-1 and AC141 or VP39, suggesting that either other nucleocapsid proteins or adaptor proteins may be required. These results further support the conclusion that microtubule transport is required for AcMNPV BV formation. IMPORTANCE In two key processes of the replication cycle of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), nucleocapsids are transported through the cell. These include (i) entry of budded virus (BV) into the host cell and (ii) egress and budding of nucleocapsids newly produced from the plasma membrane. Prior studies have shown that the entry of nucleocapsids involves the polymerization of actin to propel nucleocapsids to nuclear pores and entry into the nucleus. For the spread of infection, progeny viruses must rapidly exit the infected cells, but the mechanism by which AcMNPV nucleocapsids traverse the cytoplasm is unknown. In this study, we examined whether nucleocapsids interact with lepidopteran kinesin-1 motor molecules and are potentially carried as cargo on microtubules to the plasma membrane in AcMNPV-infected cells. This study indicates that microtubule transport is utilized for the production of budded virus. 

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