Minus Ends Research Articles

Dynamic Dissection of Dynein and Kinesin-1 Cooperatively Mediated Intercellular Transport of Porcine Epidemic Diarrhea Coronavirus along Microtubule Using Single Virus Tracking

ABSTRACT It is now clear that the intercellular transport on microtubules by dynein and kinesin-1 motors has an important role in the replication and spread of many viruses. Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded RNA virus of the Coronavirus family, which can infect swine of all ages and cause severe economic losses in the swine industry. Elucidating the molecular mechanisms of the intercellular transport of PEDV through microtubule, dynein and kinesin-1 will be crucial for understanding its pathogenesis. Here, we demonstrate that microtubule, dynein, and kinesin-1 are involved in PEDV infection and can influence PEDV fusion and accumulation in the perinuclear region but cannot affect PEDV attachment or internalization. Furthermore, we adopted a single-virus tracking technique to dynamically observe PEDV intracellular transport with five different types: unidirectional movement toward microtubule plus ends; unidirectional movement toward microtubule minus ends; bidirectional movement along the same microtubule; bidirectional movement along different microtubules and motionless state. Among these types, the functions of dynein and kinesin-1 in PEDV intercellular transport were further analyzed by single-virus tracking and found that dynein and kinesin-1 mainly transport PEDV to the minus and plus ends of the microtubules, respectively; meanwhile, they also can transport PEDV to the opposite ends of the microtubules different from their conventional transport directions and also coordinate the bidirectional movement of PEDV along the same or different microtubules through their cooperation. These results provided deep insights and references to understand the pathogenesis of PEDV as well as to develop vaccines and treatments.

Clasps Depolymerize Microtubules in a Nucleotide-Dependent Manner

CLASPs constitute a family of evolutionarily-conserved TOG-domain proteins that regulate microtubules in fundamental cellular processes including cell division, cell migration and neuronal development. CLASPs stabilize dynamically growing microtubules by suppressing catastrophe and promoting rescue, the switch-like transitions between growth and shrinkage. However, the molecular mechanisms underlying CLASP's activity are not fully understood. Previous work found that another TOG-domain protein, XMAP215/chTOG, promotes microtubule polymerization in the presence of soluble tubulin but depolymerizes microtubules in the absence of tubulin. Here, we report the surprising finding that human CLASP1 depolymerizes stabilized microtubules in the presence of GTP and GDP but not in the presence of GMPCPP nor in the no nucleotide condition. Thus, CLASP1 depolymerizes stable microtubules in a nucleotide-dependent manner. Further analyses revealed that other members of the human CLASP family (CLASP2α and CLASP2γ) also possess nucleotide-dependent depolymerase activity, as does a minimal TOG2-domain construct. Next, we demonstrate that CLASP1 depolymerizes both plus and minus microtubule ends in a nucleotide-dependent manner; this is unexpected, given that the exchangeable nucleotide site in tubulin is presumed to be buried within the polymer at the minus end. by performing single molecule dwell time analysis, we find that GTP reduces the dwell time of TOG2 at microtubule tips compared to the no nucleotide condition. Finally, we report that the depolymerase activity of CLASP1 exhibits significantly greater sensitivity to GTP than that of XMAP215/chTOG, indicating that the two TOG-domain proteins employ distinct mechanisms to depolymerize microtubules. The unanticipated finding that CLASPs possesses nucleotide-sensitive depolymerase activity provides critical mechanistic insights into the activity of an important family of microtubule regulatory proteins. This work will further our understanding of the mechanisms employed by CLASPs in regulating microtubule network dynamics and organization in cells.

Reconstitution of the recombinant human γ-tubulin ring complex.

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

Microtubule Nucleation Properties of Single Human γTurcs Investigated by TIRF Microscopy and Cryo-Electron Microscopy

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that single γTuRC complexes nucleate microtubules is a cooperative and stochastic manner. They stably cap microtubule minus ends. Nucleation is inefficient compared to microtubule elongation. A 4 Å resolution cryo-EM structure of γTuRC, combined with crosslinking-mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a ‘closed’ conformation matching the microtubule geometry. The opposite side of γTuRC is in an ‘open’, nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure. [published in Dev. Cell 53, 603-617, 2020; https://doi.org/10.1016/j.devcel.2020.04.019].

Collective Effects of Xmap215, EB1, CLASP2, and MCAK Lead to Robust Microtubule Treadmilling

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

KIF15 Supports Spermatogenesis Via Its Effects on Sertoli Cell Microtubule, Actin, Vimentin, and Septin Cytoskeletons

Throughout spermatogenesis, cellular cargoes including haploid spermatids are required to be transported across the seminiferous epithelium, either toward the microtubule (MT) plus (+) end near the basement membrane at stage V, or to the MT minus (-) end near the tubule lumen at stages VI to VIII of the epithelial cycle. Furthermore, preleptotene spermatocytes, differentiated from type B spermatogonia, are transported across the Sertoli cell blood-testis barrier (BTB) to enter the adluminal compartment. Few studies, however, have been conducted to explore the function of MT-dependent motor proteins to support spermatid transport during spermiogenesis. Herein, we examined the role of MT-dependent and microtubule plus (+) end-directed motor protein kinesin 15 (KIF15) in the testis. KIF15 displayed a stage-specific expression across the seminiferous epithelium, associated with MTs, and appeared as aggregates on the MT tracks that aligned perpendicular to the basement membrane and laid across the entire epithelium. KIF15 also tightly associated with apical ectoplasmic specialization, displaying strict stage-specific distribution, apparently to support spermatid transport across the epithelium. We used a loss-of-function approach by RNAi to examine the role of KIF15 in Sertoli cell epithelium in vitro to examine its role in cytoskeletal-dependent Sertoli cell function. It was noted that KIF15 knockdown by RNAi that reduced KIF15 expression by ~70% in Sertoli cells with an established functional tight junction barrier impeded the barrier function. This effect was mediated through remarkable changes in the cytoskeletal organization of MTs, but also actin-, vimentin-, and septin-based cytoskeletons, illustrating that KIF15 exerts its regulatory effects well beyond microtubules.

Motor Proteins and Spermatogenesis.

Unlike the intermediate filament- and septin-based cytoskeletons which are apolar structures, the microtubule (MT) and actin cytoskeletons are polarized structures in mammalian cells and tissues including the testis, most notable in Sertoli cells. In the testis, these cytoskeletons that stretch across the epithelium of seminiferous tubules and lay perpendicular to the basement membrane of tunica propria serve as tracks for corresponding motor proteins to support cellular cargo transport. These cargoes include residual bodies, phagosomes, endocytic vesicles and most notably developing spermatocytes and haploidspermatids which lack the ultrastructures of motile cells (e.g., lamellipodia, filopodia). As such,these developing germ cells require the corresponding motor proteins to facilitate theirtransport across the seminiferousepithelium duringthe epithelial cycle of spermatogenesis. Due to the polarized natures of these cytoskeletons with distinctive plus (+) and minus (-) end, directional cargo transport can take place based on the use of corresponding actin- or MT-based motor proteins. These include the MT-based minus (-) end directed motor proteins: dyneins, and the plus (+) end directed motor proteins: kinesins, as well as the actin-based motor proteins: myosins, many of which are plus (+) end directed but a few are also minus (-) end directed motorproteins. Recent studies have shown that these motor proteins are essential to support spermatogenesis. In this review, we briefly summarize and evaluate these recent findings so that this information will serve as a helpful guide for future studies and for planning functional experiments to better understand their role mechanistically in supporting spermatogenesis.

Computational modeling of dynein motor proteins at work.

Along with various experimental methods, a combination of theoretical and computational methods is essential to explore different length-scale and time-scale processes in the biological system. The functional mechanism of a dynein, an ATP-fueled motor protein, working in a multiprotein complex, involves a wide range of length/time-scale events. It generates mechanical force from chemical energy and moves on microtubules towards the minus end direction while performing a large number of biological processes including ciliary beating, intracellular material transport, and cell division. Like in the cases of other conventional motor proteins, a combination of experimental techniques including X-crystallography, cryo-electron microscopy, and single molecular assay have provided a wealth of information about the mechanochemical cycle of a dynein. Dyneins have a large and complex structural architecture and therefore, computational modeling of different aspects of a dynein is extremely challenging. As the process of dynein movement involves varying length and timescales, it demands, like in experiments, a combination of computational methods covering such a wide range of processes for the comprehensive investigation of the mechanochemical cycle. In this review article, we will summarize how the use of state-of-the-art computational methods can provide a detailed molecular understanding of the mechanochemical cycle of the dynein. We implemented all-atom molecular dynamics simulations and hybrid quantum-mechanics/molecular-mechanics simulations to explore the ATP hydrolysis mechanisms at the primary ATPase site (AAA1) of dynein. To investigate the large-scale conformational changes we employed coarse-grained structure-based molecular dynamics simulations to capture the domain motions. Here we explored the conformational changes upon binding of ATP at AAA1, nucleotide state-dependent regulation of the mechanochemical cycle, and inter-head coordination by inter-head tension. Additionally, implementing a phenomenological theoretical model we explore the force-dependent detachment rate of a motorhead from the microtubule and the principle of multi-dynein cooperation during cargo transport.

The Ways of Actin: Why Tunneling Nanotubes Are Unique Cell Protrusions.

Actin remodeling is at the heart of the response of cells to external or internal stimuli, allowing a variety of membrane protrusions to form. Fifteen years ago, tunneling nanotubes (TNTs) were identified, bringing a novel addition to the family of actin-supported cellular protrusions. Their unique property as conduits for cargo transfer between distant cells emphasizes the unique nature of TNTs among other protrusions. While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood. In this review, we discuss the role of several actin regulators in the formation of TNTs and suggest potential players based on their comparison with other actin-based protrusions. New perspectives for discovering a distinct TNT formation pathway would enable us to target them in treating the increasing number of TNT-involved pathologies.

Mechanobiology of the Mitotic Spindle.

The mitotic spindle is a microtubule-based assembly that separates the chromosomes during cell division. As the spindle is basically a mechanical micro machine, the understanding of its functioning is constantly motivating the development of experimental approaches based on mechanical perturbations, which are complementary to and work together with the classical genetics and biochemistry methods. Recent data emerging from these approaches in combination with theoretical modeling led to novel ideas and significant revisions of the basic concepts in the field. In this Perspective, we discuss the advances inthe understanding of spindle mechanics, focusing on microtubule forces that control chromosome movements.

Cargo-Mediated Activation of Cytoplasmic Dynein in vivo.

Cytoplasmic dynein-1 is a minus-end-directed microtubule motor that transports a variety of cargoes including early endosomes, late endosomes and other organelles. In many cell types, dynein accumulates at the microtubule plus end, where it interacts with its cargo to be moved toward the minus end. Dynein binds to its various cargoes via the dynactin complex and specific cargo adapters. Dynactin and some of the coiled-coil-domain-containing cargo adapters not only link dynein to cargo but also activate dynein motility, which implies that dynein is activated by its cellular cargo. Structural studies indicate that a dynein dimer switches between the autoinhibited phi state and an open state; and the binding of dynactin and a cargo adapter to the dynein tails causes the dynein motor domains to have a parallel configuration, allowing dynein to walk processively along a microtubule. Recently, the dynein regulator LIS1 has been shown to be required for dynein activation in vivo, and its mechanism of action involves preventing dynein from switching back to the autoinhibited state. In this review, we will discuss our current understanding of dynein activation and point out the gaps of knowledge on the spatial regulation of dynein in live cells. In addition, we will emphasize the importance of studying a complete set of dynein regulators for a better understanding of dynein regulation in vivo.

Dynamic Instability from Non-equilibrium Structural Transitions on the Energy Landscape of Microtubule.

Microtubules are the backbone of the cytoskeleton and vital to numerous cellular processes. The central dogma of microtubules is that all their functions are driven by dynamic instability, but its mechanism has remained unresolved for over 30 years because of conceptual difficulties inherent in the dominant GTP-cap framework. We present a physically rigorous structural mechanochemical model: dynamic instability is driven by non-equilibrium transitions between the bent (B), straight (S), and curved (C) forms of tubulin monomers and longitudinal interfaces in the two-dimensional lattice of microtubule. All the different phenomena (growth, shortening, catastrophe, rescue, and pausing) are controlled by the kinetic pathways for B↔S↔C transitions and corresponding energy landscapes. Different kinetics at minus end are due to different B↔S↔C pathways imposed by the polarity of microtubule lattice. This model enables us to reproduce all the observed phenomena of dynamic instability of purified tubulins in kinetic simulations.

Suppression of Patronin deficiency by altered Hippo signaling in Drosophila organ development

The microtubule network is crucial for cell structure and function. Patronin is a conserved protein involved in protecting the minus end of microtubules. Conversely, Klp10A is a kinesin-like microtubule depolymerase. Here we report the role of Drosophila Patronin and Klp10A for cell survival in developing organs. Loss of Patronin reduces the size of organs by activation of a caspase in imaginal discs. Reduced wing by Patronin RNAi is suppressed by knockdown of Spastin (Spas) but not Katanin 60, suggesting that Patronin is inhibitory to the severing function of Spas at the minus end. Patronin RNAi phenotype is also recovered by overexpressing Death-associated inhibitor of apoptosis 1 (Diap1), a Yorkie target gene. Heterozygote mutations in Hippo pathway genes, including hippo and warts (wts), suppress the Patronin RNAi wing phenotypes. Furthermore, Patronin physically interacts with Merlin and Expanded while reducing their function. Patronin and Klp10A antagonistically regulate their levels. Wing phenotypes of Patronin RNAi are rescued by knockdown of Klp10A, consistent with their antagonistic interaction. Klp10A overexpression also causes organ size reduction that is partially suppressed by Diap1 overexpression or wts heterozygote mutation. Taken together, this study suggests that the antagonistic interaction between Patronin and Klp10A is required for controlling cell survival and organ size by modulating microtubule stability and Hippo components.

Collective effects of XMAP215, EB1, CLASP2, and MCAK lead to robust microtubule treadmilling

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure.

At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.

SummaryThe γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a “closed” conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an “open,” nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

Association between aberrant dynein cytoplasmic1 light intermediate chain1 expression levels, mucins and chemosensitivity in colorectal cancer.

Dynein transport along the cytoskeletal microtubules towards the minus end is essential for cell division, cell migration and other basic cellular functions. Dynein cytoplasmic 1 light intermediate chain 1 (DYNC1LI1) has been previously associated with pancreatic ductal adenocarcinoma, hepatocellular carcinoma and prostate cancer. Cytoskeletal structures are involved in the regulation of the mucosal barrier integrity. Thus, improving our understanding of the molecular mechanisms that regulate the mucosal barrier is critical for cancer management and treatment. The present study aimed to investigate DYNC1LI1 expression in colorectal cancer (CRC) tissues. The American Joint Committee on Cancer Stage II CRC cell line LS 174T was used to determine the association between the cellular expression levels of DYNC1LI1 and different types of mucin (MUC) by reverse transcription-quantitative PCR. The role of DYNC1LI1 in cell chemosensitivity and proliferation was also evaluated in the presence of the DNA analog 5-fluorouracil (5-FU) or the platinum-based drug, oxaliplatin by the MTT assay. LS 174T cells with decreased expression levels of DYNC1LI1 were discovered to be more sensitive to 5-FU compared with LS 174T cells with endogenous DYNC1LI1 expression levels. Moreover, LS 174T cells transfected with short hairpin RNA targeting DYNC1LI1 were associated with low MUC1 and high MUC2, MUC4 and MUC5AC expression levels. Notably, the CRC cells with low MUC1 expression levels and high expression levels of the other MUCs (MUC2, MU4 and MUC5AC) were shown to benefit from 5-FU treatment. In conclusion, the findings of the present study have suggested that DYNC1LI1 expression may be significantly associated with MUC expression levels and may be used to predict the chemotherapeutic efficiency. However, additional functional studies and clinical reports are required for an improved understanding of the significance of these molecular interactions in tumorigenesis.

How Cytoplasmic Dynein Couples ATP Hydrolysis Cycle to Diverse Stepping Motions: Kinetic Modeling

Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein exhibits various bipedal stepping motions: inchworm and hand-over-hand motions, as well as nonalternating steps of one head. However, the molecular basis to achieve such diverse stepping manners remains unclear because of the lack of an experimental method to observe stepping and the ATPase reaction of dynein simultaneously. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and perform Gillespie Monte Carlo simulations that qualitatively reproduce most experimental data obtained to date. The model represents the status of each motor domain as five states according to conformation and nucleotide- and microtubule-binding conditions of the domain. In addition, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, similarly to experimental data. The model reproduced key experimental motility-related properties, including velocity and run length, as functions of the ATP concentration and external force, therefore providing a plausible explanation of how dynein achieves various stepping manners with explicit characterization of nucleotide states. Our model highlights the uniqueness of dynein in the coupling of ATPase with its movement during both inchworm and hand-over-hand stepping.

Microtubule Minus-End Binding Protein CAMSAP2 and Kinesin-14 Motor KIFC3 Control Dendritic Microtubule Organization

SummaryNeuronal dendrites are characterized by an anti-parallel microtubule organization. The mixed oriented microtubules promote dendrite development and facilitate polarized cargo trafficking; however, the mechanism that regulates dendritic microtubule organization is still unclear. Here, we found that the kinesin-14 motor KIFC3 is important for organizing dendritic microtubules and to control dendrite development. The kinesin-14 motor proteins (Drosophila melanogaster Ncd, Saccharomyces cerevisiae Kar3, Saccharomyces pombe Pkl1, and Xenopus laevis XCTK2) are characterized by a C-terminal motor domain and are well described to organize the spindle microtubule during mitosis using an additional microtubule binding site in the N terminus [1, 2, 3, 4]. In mammals, there are three kinesin-14 members, KIFC1, KIFC2, and KIFC3. It was recently shown that KIFC1 is important for organizing axonal microtubules in neurons, a process that depends on the two microtubule-interacting domains [5]. Unlike KIFC1, KIFC2 and KIFC3 lack the N-terminal microtubule binding domain and only have one microtubule-interacting domain, the motor domain [6, 7]. Thus, in order to regulate microtubule-microtubule crosslinking or sliding, KIFC2 and KIFC3 need to interact with additional microtubule binding proteins to connect two microtubules. We found that KIFC3 has a dendrite-specific distribution and interacts with microtubule minus-end binding protein CAMSAP2. Depletion of KIFC3 or CAMSAP2 results in increased microtubule dynamics during dendritic development. We propose a model in which CAMSAP2 anchors KIFC3 at microtubule minus ends and immobilizes microtubule arrays in dendrites.

Interaction between Autonomous and Microtubule Guidance Systems Controls Cellulose Synthase Trajectories.

The organization of cellulose microfibrils is critical for the strength and growth of plant cell walls. Microtubules have been shown to play a key role in controlling microfibril organization by guiding cellulose synthase complexes [1-4]. However, cellulose synthase trajectories can be maintained when microtubules are removed by drugs, suggesting a separate guidance mechanism is also at play [1, 5, 6]. By slowing down microtubule dynamics, we reveal such a mechanism by showing that cellulose synthase complexes can interact with the trails left by other complexes, causing them to follow the trails or disappear. The stability of the trails, together with the sensitivity of their directions to cellulase treatment, indicates they most likely reflect nascent cellulose microfibrils. Over many hours, this autonomous mechanism alone can lead to a change in the dominant orientation of cellulose synthase trajectories. However, the mechanism can be overridden by the microtubule guidance system. Our findings suggest a dual guidance model, in which an autonomous system, involving interaction between cellulose synthases and microfibrils, can maintain aligned cellulose synthase trajectories, while a microtubule guidance system allows alignments to be steered by environmental and developmental cues.