A study of the fruit fly Drosophila reveals that receptor-mediated yolk uptake is not merely a nutrient storage process for future embryogenesis, but is also required for localization of Oskar mRNA and cortical anchorage of germ plasm components in the oocyte during oogenesis.

Proteins of the ezrin, radixin, and moesin (ERM) family control cell and tissue morphogenesis. We previously reported that moesin, the only ERM in Drosophila, controls mitotic morphogenesis and epithelial integrity. We also found that the Pp1-87B phosphatase dephosphorylates moesin, counteracting its activation by the Ste20-like kinase Slik. To understand how this signaling pathway is itself regulated, we conducted a genome-wide RNAi screen, looking for new regulators of moesin activity. We identified that Slik is a new member of the striatin-interacting phosphatase and kinase complex (STRIPAK). We discovered that the phosphatase activity of STRIPAK reduces Slik phosphorylation to promote its cortical association and proper activation of moesin. Consistent with this finding, inhibition of STRIPAK phosphatase activity causes cell morphology defects in mitosis and impairs epithelial tissue integrity. Our results implicate the Slik–STRIPAK complex in the control of multiple morphogenetic processes.

Arp2/3-nucleated actin filaments drive crawling motility and phagocytosis in animal cells and slime molds. In this issue, Velle and Fritz-Laylin (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202007158) now show that Naegleria gruberi, belonging to a lineage that diverged from opisthokonts around a billion years ago, uses similar mechanisms to crawl and phagocytose bacteria.

Much of our understanding of actin-driven phenotypes in eukaryotes has come from the “yeast-to-human” opisthokont lineage and the related amoebozoa. Outside of these groups lies the genus Naegleria, which shared a common ancestor with humans >1 billion years ago and includes the “brain-eating amoeba.” Unlike nearly all other known eukaryotic cells, Naegleria amoebae lack interphase microtubules; this suggests that actin alone drives phenotypes like cell crawling and phagocytosis. Naegleria therefore represents a powerful system to probe actin-driven functions in the absence of microtubules, yet surprisingly little is known about its actin cytoskeleton. Using genomic analysis, microscopy, and molecular perturbations, we show that Naegleria encodes conserved actin nucleators and builds Arp2/3–dependent lamellar protrusions. These protrusions correlate with the capacity to migrate and eat bacteria. Because human cells also use Arp2/3–dependent lamellar protrusions for motility and phagocytosis, this work supports an evolutionarily ancient origin for these processes and establishes Naegleria as a natural model system for studying microtubule-independent cytoskeletal phenotypes.

Tau protein in vitro can undergo liquid–liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.

Natural killer (NK) cell–mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell–mediated killing.

The mechanisms by which TP53, the most frequently mutated gene in human cancer, suppresses tumorigenesis remain unclear. p53 modulates various cellular processes, such as apoptosis and proliferation, which has led to distinct cellular mechanisms being proposed for p53-mediated tumor suppression in different contexts. Here, we asked whether during tumor suppression p53 might instead regulate a wide range of cellular processes. Analysis of mouse and human oncogene-expressing wild-type and p53-deficient cells in physiological oxygen conditions revealed that p53 loss concurrently impacts numerous distinct cellular processes, including apoptosis, genome stabilization, DNA repair, metabolism, migration, and invasion. Notably, some phenotypes were uncovered only in physiological oxygen. Transcriptomic analysis in this setting highlighted underappreciated functions modulated by p53, including actin dynamics. Collectively, these results suggest that p53 simultaneously governs diverse cellular processes during transformation suppression, an aspect of p53 function that would provide a clear rationale for its frequent inactivation in human cancer.

The epithelium that lines our intestine has the important task of taking up nutrients, while also providing a barrier against pathogens. The intestinal epithelium performs these different tasks by having specialized cell types; enterocytes take up nutrients whereas goblet cells are in charge of producing a mucus layer. In addition, goblet cells can be stimulated to make special antimicrobial proteins. This occurs in response to cues called cytokines that come from immune cells, which are able to detect and act on the presence of pathogens such as bacteria or parasitic worms. In this study, we found that LSD1, an enzyme that controls gene expression, was important for goblet cells. Mice that lacked LSD1 specifically in their intestinal epithelium were unable to respond to cytokines and could not defend themselves against bacterial and parasitic infections. In part, we also made use of a specific inhibitor against the enzyme activity of LSD1. This inhibitor also blocked goblet cell differentiation and goblet-cell specific antimicrobial responses to cytokines. We are thus able to manipulate epithelial responses, which may be an important tool in the future to treat patients with infectious diseases.

The kinesin-3 motor KIF1A functions in neurons, where its fast and superprocessive motility facilitates long-distance transport, but little is known about its force-generating properties. Using optical tweezers, we demonstrate that KIF1A stalls at an opposing load of ~3 pN but more frequently detaches at lower forces. KIF1A rapidly reattaches to the microtubule to resume motion due to its class-specific K-loop, resulting in a unique clustering of force generation events. To test the importance of neck linker docking in KIF1A force generation, we introduced mutations linked to human neurodevelopmental disorders. Molecular dynamics simulations predict that V8M and Y89D mutations impair neck linker docking. Indeed, both mutations dramatically reduce the force generation of KIF1A but not the motor’s ability to rapidly reattach to the microtubule. Although both mutations relieve autoinhibition of the full-length motor, the mutant motors display decreased velocities, run lengths, and landing rates and delayed cargo transport in cells. These results advance our understanding of how mutations in KIF1A can manifest in disease.

Pavarotti, the Drosophila MKLP1 orthologue, is a kinesin-like protein that works with Tumbleweed (MgcRacGAP) as the centralspindlin complex. This complex is essential for cytokinesis, where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. We previously showed that Tumbleweed and Pebble are required for the cell wound repair process. Here, we show that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are all used during this cellular repair, each has a unique localization pattern and knockdown phenotype, demonstrating centralspindlin-independent functions. Surprisingly, we find that the classically microtubule-associated Pavarotti binds directly to actin in vitro and in vivo and has a noncanonical role directly regulating actin dynamics. Finally, we demonstrate that this actin regulation by Pavarotti is not specific to cellular wound repair but is also used in normal development.

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm), very little is known about cytoplasmic cilia assembly. Here, we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme.

Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.

Microtubule (MT) plus-end tracking proteins (+TIPs) are central players in the coordination between the MT and actin cytoskeletons in growth cones (GCs) during axon guidance. The +TIP Navigator-1 (NAV1) is expressed in the developing nervous system, yet its neuronal functions remain poorly elucidated. Here, we report that NAV1 controls the dynamics and motility of the axonal GCs of cortical neurons in an EB1-dependent manner and is required for axon turning toward a gradient of netrin-1. NAV1 accumulates in F-actin–rich domains of GCs and binds actin filaments in vitro. NAV1 can also bind MTs independently of EB1 in vitro and crosslinks nonpolymerizing MT plus ends to actin filaments in axonal GCs, preventing MT depolymerization in F-actin–rich areas. Together, our findings pinpoint NAV1 as a key player in the actin–MT crosstalk that promotes MT persistence at the GC periphery and regulates GC steering. Additionally, we present data assigning to NAV1 an important role in the radial migration of cortical projection neurons in vivo.

Functional and structural distinct axonal and dendritic compartments demand specific regulation of mitochondrial distribution. While most of previous studies examined mitochondrial transport in cultured neurons or at a certain segment of axon, we genetically ablated five motors and four regulators and study their roles in a sensory neuron that has two microtubule polarity distinct dendrites and an axon. We showed that dynein regulates mitochondrial distribution in neurites based on microtubule polarity regardless of axon or dendrite, while KIF5, but not the other kinesin motors, mediates an axon-specific mitochondrial transport. We surprisingly found that CRMP/UNC-33 is critical for axonal mitochondria and it supports a stable microtubule population to form a spindle-like structure in neuronal cell body as tracks for trafficking. These results indicate that CRMP/UNC-33 promotes high-order organization of microtubule bundles in neurons, which are cells without a clear centralized microtubule organization center but still possess highly organized microtubule bundles. As defects of mitochondrial trafficking are implicated in most of the neurodegeneration diseases, this study manifests specific roles of motors and regulators in distinct axonal and dendritic compartments, and it further reveals how the microtubule network forms in the neuronal cell body.

Kinetochores, the site on the chromosomes to which microtubules attach driving the separation and segregation of replicated sister chromosomes, have been viewed as essential for proper cell division and accurate transmission of chromosomes into daughter cells. However previous studies demonstrated that sister chromosomes lacking kinetochores (acentrics) often undergo separation, segregation and transmission. Here we demonstrate that sister acentrics are held together through DNA intertwining. We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers. We found that acentric sister chromatids display unique patterns of separation using mechanisms independent of the kinetochore. Additionally, we identified the specific microtubule-associated proteins required for the successful mitotic transmission of acentric chromosomes to daughter cells. These studies reveal unsuspected, distinct forces that likely act on all chromosomes during mitosis independent of kinetochore-microtubule attachments.

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

Intracellular pathogens modulate host cell actin cytoskeleton by secreting an array of effector molecules to ensure their cell invasion and intracellular survival. The zoonotic pathogen Bartonella spp trigger massive F-actin polymerization of host cells resulting the internalization of large bacterial aggregates (called “invasome” structure), which is dependent on a functional VirB/VirD4 type IV secretion system (T4SS) and its translocated Bep effector proteins. Here, we have used cell infection and ectopic expression assay to identify that Bartonella T4SS effector BepC induces stress fiber formation in infected host cells. However, BepC also disrupts the balance of stress fiber formation and focal adhesion maturation, and eventually causes cell fragmentation. Using immunoprecipitation and RNAi approaches, we identify GEF-H1 is the host factor targeted by BepC. Interaction with BepC induces the release of GEF-H1 from microtubules to plasma membrane and subsequently activates RhoA-ROCK to induce stress fiber formation. These findings shed light on our understanding of how Bartonella invade host cell and establish infection.

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.

Motile cilia in various kinds of tissues and cell-types drive fluid flow over epithelia or facilitate cellular locomotion. There are two types of motile cilia. Motile cilia with a 9+2 configuration of microtubules are found on tracheal epithelial cells and brain ependymal cells, and exhibit planar beating with effective and recovery strokes. On the other hand, 9+0 motile cilia are found in the embryonic node, show rotational movement and are involved in establishing left-right asymmetry of visceral organs. However, it is not well understood how these two types of motile cilia exhibit their characteristic motion patterns. We have uncovered distinct roles and subcellular localization of the CFAP53 protein in 9+0 versus the 9+2 motile cilia of the mouse. Our data provide novel insights into the molecular basis of motility differences that characterize these two types of mammalian motile cilia.

Precise control of cytoskeleton dynamics and its tight coordination with chromosomal events are key to cell division. This is exemplified by formation of the spindle and execution of cytokinesis after nuclear division. Here, we reveal that the central cell cycle regulator CYCLIN DEPENDENT KINASE A;1 (CDKA;1), the Arabidopsis homologue of Cdk1 and Cdk2, partially in conjunction with CYCLIN B3;1 (CYCB3;1), is a key regulator of the microtubule cytoskeleton in meiosis. For full CDKA;1 activity, the function of three redundantly acting CDK-activating kinases (CAKs), CDKD;1, CDKD;2, and CDKD;3, is necessary. Progressive loss of these genes in combination with a weak loss-of-function mutant in CDKA;1 allowed a fine-grained dissection of the requirement of cell-cycle kinase activity for meiosis. Notably, a moderate reduction of CDKA;1 activity converts the simultaneous cytokinesis in Arabidopsis, i.e., one cytokinesis separating all four meiotic products concurrently into two successive cytokineses with cell wall formation after the first and second meiotic division, as found in many monocotyledonous species.

The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. Here we report a direct view of Drosophila centriolar proteins at ∼50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1–Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135–Ana1–Asterless complex. Together, our results provide a spatiotemporal map of the centriole core and implications of how the structure might be built.

The microtubule cytoskeleton serves as a dynamic structural framework for mitosis in eukaryotic cells. TANGLED1 (TAN1) is a microtubule-binding protein that localizes to the division site and mitotic microtubules and plays a critical role in division plane orientation in plants. Here, in vitro experiments demonstrate that TAN1 directly binds microtubules, mediating microtubule zippering or end-on microtubule interactions, depending on their contact angle. Maize tan1 mutant cells improperly position the preprophase band (PPB), which predicts the future division site. However, cell shape–based modeling indicates that PPB positioning defects are likely a consequence of abnormal cell shapes and not due to TAN1 absence. In telophase, colocalization of growing microtubules ends from the phragmoplast with TAN1 at the division site suggests that TAN1 interacts with microtubule tips end-on. Together, our results suggest that TAN1 contributes to microtubule organization to ensure proper division plane orientation.

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.

Giant ankyrin-B (gAnkB) is a 440-kD neurospecific ankyrin-B isoform and a high-confidence target for autism mutations. gAnkB suppresses axon branching through coordination of cortical microtubules, and autism-related mutation of gAnkB results in ectopic neuronal connectivity. We identified a bipartite motif from gAnkB, which bundles and avidly binds to microtubules in vitro. This motif is composed of a module of 15 tandem repeats followed by a short, conserved fragment also found in giant ankyrin-G (BG-box). Combination of these two parts synergistically increases microtubule-binding avidity. Transfection of astrocytes (which lack gAnkB) with WT gAnkB resulted in prominent bundling of microtubules, which did not occur with mutant gAnkB with impaired microtubule-binding activity. Similarly, rescue of gAnkB-deficient neurons with WT gAnkB suppressed axonal branching and invasion of EB3-tagged microtubules into filopodia, which did not occur with the same mutant gAnkB. Together, these findings demonstrate that gAnkB suppresses axon collateral branching and prevents microtubule invasion of nascent axon branches through direct interaction with microtubules.

A wide variety of bacterial pathogens evolved numerous virulence factors to subvert cellular processes in support of a successful infection process. Likewise, bacteria of the genus Bartonella translocate a cocktail of effector proteins (Beps) via a type-IV-secretion system into infected cells in order to interfere with host signaling processes involved in cytoskeletal dynamics, apoptosis control, and innate immune responses. In this study, we demonstrate that BepC triggers actin stress fiber formation and a linked cell fragmentation phenotype resulting from distortion of rear-end retraction during cell migration. The ability of BepC to induce actin stress fiber formation is directly associated with its ability to bind GEF-H1, an activator of the RhoA pathway that is sequestered in an inactive state when bound to microtubules but becomes activated upon release to the cytoplasm. Our findings suggest that BepC is anchored via its BID domain to the plasma membrane where it recruits GEF-H1 via its FIC domain, eventually activating the RhoA/ROCK signaling pathway and leading to stress fiber formation.

Different types of neurons have different sizes and shapes that are tailored to their particular functions. In the fruit fly larva, a set of sensory neurons called class IV da neurons have highly branched trees of dendrites that cover the epidermis to sense potentially harmful stimuli. Neurons whose dendrites completely fill the territory they sample are also found in zebrafish, worms, mice and humans. We show that an RNA-binding protein called Fne plays an important role in coordinating different contributions to dendrite branching in class IV da neurons by impacting the organization of the cytoskeleton within the neuron and the ability of the dendrite to contact the substratum outside of it. The identification of mRNAs that code for cytoskeleton regulators and adhesive proteins as targets of Fne using a genome-wide approach further supports these results. While the ability of Fne to exert control over such proteins is crucial to generating the space-filling growth of neurons, it can be deleterious if not properly employed, such as when the homologs of Fne are expressed in cancer cells.

The maintenance of axons for the lifetime of an organism requires an axonal cytoskeleton that is robust but also flexible to adapt to mechanical challenges and to support plastic changes of axon morphology. Furthermore, cytoskeletal organization has to adapt to axons of dramatically different dimensions, and to their compartment-specific requirements in the axon initial segment, in the axon shaft, at synapses or in growth cones. To understand how the cytoskeleton caters to these different demands, this review summarizes five decades of electron microscopic studies. It focuses on the organization of microtubules and neurofilaments in axon shafts in both vertebrate and invertebrate neurons, as well as the axon initial segments of vertebrate motor- and interneurons. Findings from these ultrastructural studies are being interpreted here on the basis of our contemporary molecular understanding. They strongly suggest that axon architecture in animals as diverse as arthropods and vertebrates is dependent on loosely cross-linked bundles of microtubules running all along axons, with only minor roles played by neurofilaments.

The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170’s capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.