A Force-Induced Directional Switch of a Molecular Motor Enables Parallel Microtubule Bundle Formation.

Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.

Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1.

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

Efficient retinal ganglion cells transduction by retro-orbital venous sinus injection of AAV-PHP.eB in mature mice.

Gene therapy is one of the strategies that may reduce or reverse progressive neurodegeneration in retinal neurodegenerative diseases. However, efficiently delivering transgenes to retinal ganglion cells (RGCs) remains hard to achieve. In this study, we innovatively investigated transduction efficiency of adeno-associated virus (AAV)-PHP.eB in murine RGCs by retro-orbital venous sinus injection. Five doses of AAV-PHP.eB-EGFP were retro-orbitally injected in venous sinus in adult C57/BL6J mice. Two weeks after administration, RGCs transduction efficiency was quantified by retinal flat-mounts and frozen section co-labeling with RGCs marker Rbpms. In addition, safety of this method was evaluated by RGCs survival rate and retinal morphology. To conform efficacy of this new method, AAV-PHP.eB-CNTF was administrated into mature mice through single retro-orbital venous injection after optic nerve crush injury to evaluate axonal elongation. Results indicated that AAV- PHP.eB readily crossed the blood-retina barrier and was able to transduce more than 90% of RGCs when total dose of virus reached 5 × 1010 vector genomes (vg). Moreover, this technique did not affect RGCs survival rate and retinal morphology. Furthermore, retro-orbital venous delivery of AAV-PHP.eB-CNTF effectively transduced RGCs, robustly promoted axonal regeneration after optic nerve crush injury. Thus, novel AAV-PHP.eB retro-orbital injection provides a minimally invasive and efficient route for transgene delivery in treatment of retinal neurodegenerative diseases.

Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Septum-associated microtubule organizing centers within conidia support infectious development by the blast fungus Magnaporthe oryzae.

Cytoplasmic microtubule arrays play important and diverse roles within fungal cells, including serving as molecular highways for motor-driven organelle motility. While the dynamic plus ends of cytoplasmic microtubules are free to explore the cytoplasm through their stochastic growth and shrinkage, their minus ends are nucleated at discrete organizing centers, composed of large multi-subunit protein complexes. The location and composition of these microtubule organizing centers varies depending on genus, cell type, and in some instances cell-cycle stage. Despite their obvious importance, our understanding of the nature, diversity, and regulation of microtubule organizing centers in fungi remains incomplete. Here, using three-color fluorescence microscopy based live-cell imaging, we investigate the organization and dynamic behavior of the microtubule cytoskeleton within infection-related cell types of the filamentous fungus,Magnaporthe oryzae, a highly destructive pathogen of rice and wheat. We provide data to support the idea that cytoplasmic microtubules are nucleated at septa, rather than at nuclear spindle pole bodies, within the three-celled blast conidium, and provide new insight into remodeling of the microtubule cytoskeleton during nuclear division and inheritance. Lastly, we provide a more complete picture of the architecture and subcellular organization of the prototypical blast appressorium, a specialized pressure-generating cell type used to invade host tissue. Taken together, our study provides new insight into microtubule nucleation, organization, and dynamics in specialized and differentiated fungal cell types.

Validation and classification of RNA binding proteins identified by mRNA interactome capture.

RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

The microtubule end-binding affinity of EB1 is enhanced by a dimeric organization that is susceptible to phosphorylation.

In cells, microtubule dynamics are regulated by plus-end tracking proteins (+TIPs). End-binding protein 1 (EB1, also known as MAPRE1) acts as a master regulator of +TIP networks by targeting the growing ends of microtubules and recruiting other factors. However, the molecular mechanism underlying high-affinity binding of EB1 to microtubule ends remains an open area of research. Using single-molecule imaging, we show that the end-binding kinetics of EB1 change when the polymerization and hydrolysis rates of tubulin dimers are altered, confirming that EB1 binds to GTP-tubulin and/or GDP-Pi-tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than that of monomeric EB1 mutants, suggesting that both calponin homology domains present in the EB1 dimer contribute to end binding. Introduction of phosphomimetic mutations into the EB1 linker domain weakens the end-binding affinity and confers a more curved conformation on the EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for its end-binding affinity. Taken together, our results provide insights into how the high-affinity end-binding of EB1 is achieved and how this activity may be regulated in cells.

Imaging mitotic processes in three dimensions with lattice light-sheet microscopy.

There are few technologies that can capture mitotic processes occurring in three-dimensional space with the desired spatiotemporal resolution. Due to such technical limitations, our understanding of mitosis, which has been studied since the early 1880s, is still incomplete with regard to mitotic processes and their regulatory mechanisms at a molecular level. A recently developed high-resolution type of light-sheet microscopy, lattice light-sheet microscopy (LLSM), has achieved unprecedented spatiotemporal resolution scans of intracellular spaces at the whole-cell level. This technology enables experiments that were not possible before (e.g., tracking of growth of every spindle microtubule end and discrimination of individual chromosomes in living cells), thus providing a new avenue for the analysis of mitotic processes. Herein, principles of LLSM technology are introduced, as well as experimental techniques that became possible with LLSM. In addition, issues remaining to be solved for use of this technology in mitosis research, big image data problems, are presented to help guide mitosis research into a new era.

Roles of TOG and jelly-roll domains of centrosomal protein CEP104 in its functions in cilium elongation and Hedgehog signaling.

Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies.

NC1-peptide regulates spermatogenesis through changes in cytoskeletal organization mediated by EB1.

During the epithelial cycle of spermatogenesis, different sets of cellular events take place across the seminiferous epithelium in the testis. For instance, remodeling of the blood-testis barrier (BTB) that facilitates the transport of preleptotene spermatocytes across the immunological barrier and the release of sperms at spermiation take place at the opposite ends of the epithelium simultaneously at stage VIII of the epithelial cycle. These cellular events are tightly coordinated via locally produced regulatory biomolecules. Studies have shown that collagen α3 (IV) chains, a major constituent component of the basement membrane, release the non-collagenous (NC) 1 domain, a 28-kDa peptide, designated NC1-peptide, from the C-terminal region, via the action of MMP-9 (matrix metalloproteinase 9). NC1-peptide was found to be capable of inducing BTB remodeling and spermatid release across the epithelium. As such, the NC1-peptide is an endogenously produced biologically active peptide which coordinates these cellular events across the epithelium in stage VIII tubules. Herein, we used an animal model, wherein NC1-peptide cloned into the pCI-neo mammalian expression vector was overexpressed in the testis, to better understanding the molecular mechanism by which NC1-peptide regulated spermatogenic function. It was shown that NC1-peptide induced considerable downregulation on a number of cell polarity and planar cell polarity (PCP) proteins, and studies have shown these polarity and PCP proteins modulate spermatid polarity and adhesion via their effects on microtubule (MT) and F-actin cytoskeletal organization across the epithelium. More important, NC1-peptide exerted its effects by downregulating the expression of microtubule (MT) plus-end tracking protein (+TIP) called EB1 (end-binding protein 1). We cloned the full-length EB1 cDNA for its overexpression in the testis, which was found to block the NC1-peptide-mediated disruptive effects on cytoskeletal organization in Sertoli cell epithelium and pertinent Sertoli cell functions. These findings thus illustrate that NC1-peptide is working in concert with EB1 to support spermatogenesis.

Current Management and New Developments in the Treatment of Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous hematological neoplasm with a wide range of clinical presentations from isolated anemia to pancytopenia and propensity to transform to acute myeloid leukemia. MDS is characterized by morphologic bone marrow dysplasia and ineffective hematopoiesis resulting from a range of cytogenetic abnormalities and somatic gene mutations. Disease management varies from observation alone for low-risk disease to hypomethylating agents and hematopoietic cell transplantation (HCT) for higher risk disease. In this chapter, we review the classification, risk stratification, and optimal management of patients with MDS.

EB1 Is Essential for Spindle Formation and Chromosome Alignment During Oocyte Meiotic Maturation in Mice.

The cytoskeleton plays an orchestrating role in polarized cell growth. Microtubules (MTs) not only play critical roles in chromosome alignment and segregation but also control cell shape, division, and motility. A member of the plus-end tracking proteins, end-binding protein 1 (EB1), regulates MT dynamics and plays vital roles in maintaining spindle symmetry and chromosome alignment during mitosis. However, the role of EB1 in mouse oocyte meiosis remains unknown. Here, we examined the localization patterns and expression levels of EB1 at different stages. EB1 protein level was found to be stable during meiosis. EB1 mainly localized along the spindle and had a similar localization pattern as that of α-tubulin. The EB1 protein was degraded with a Trim-Away method, and the results were further confirmed with western blotting and immunofluorescence. At 12 h of culture after EB1 knockdown (KD), a reduced number of mature MII oocytes were observed. EB1 KD led to spindle disorganization, chromosome misalignment, and missegregation; ß-catenin protein binds to actin via the adherens junctional complex, which was significantly reduced in the EB1 KD oocytes. Collectively, we propose that the impairment of EB1 function manipulates spindle formation, thereby promoting chromosomal loss, which is expected to fuel aneuploidy and possibly fertilization failure.

Mapping multivalency in the CLIP-170-EB1 microtubule plus-end complex.

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule plus-end factor that links vesicles to microtubules and recruits the dynein-dynactin complex to microtubule plus ends. CLIP-170 plus-end localization is end binding 1 (EB1)-dependent. CLIP-170 contains two N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domains flanked by serine-rich regions. The CAP-Gly domains are known EB1-binding domains, and the serine-rich regions have also been implicated in CLIP-170's microtubule plus-end localization mechanism. However, the determinants in these serine-rich regions have not been identified. Here we elucidated multiple EB1-binding modules in the CLIP-170 N-terminal region. Using isothermal titration calorimetry and size-exclusion chromatography, we mapped and biophysically characterized these EB1-binding modules, including the two CAP-Gly domains, a bridging SXIP motif, and a unique array of divergent SXIP-like motifs located N-terminally to the first CAP-Gly domain. We found that, unlike the EB1-binding mode of the CAP-Gly domain in the dynactin-associated protein p150Glued, which dually engages the EB1 C-terminal EEY motif as well as the EB homology domain and sterically occludes SXIP motif binding, the CLIP-170 CAP-Gly domains engage only the EEY motif, enabling the flanking SXIP and SXIP-like motifs to bind the EB homology domain. These multivalent EB1-binding modules provided avidity to the CLIP-170-EB1 interaction, likely clarifying why CLIP-170 preferentially binds EB1 rather than the α-tubulin C-terminal EEY motif. Our finding that CLIP-170 has multiple non-CAP-Gly EB1-binding modules may explain why autoinhibition of CLIP-170 GAP-Gly domains does not fully abrogate its microtubule plus-end localization. This work expands our understanding of EB1-binding motifs and their multivalent networks.

Microtubule-associated tumor suppressors as prognostic biomarkers in breast cancer.

PURPOSE: Breast cancer is the most common malignancy in women worldwide. Although important therapeutic progress was achieved over the past decade, this disease remains a public health problem. In light of precision medicine, the identification of new prognostic biomarkers in breast cancer is urgently needed to stratify populations of patients with poor clinical outcome who may benefit from new personalized therapies. The microtubule cytoskeleton plays a pivotal role in essential cellular functions and is an interesting target for cancer therapy. Microtubule assembly and dynamics are regulated by a wide range of microtubule-associated proteins (MAPs), some of which have oncogenic or tumor suppressor effects in breast cancer. RESULTS: This review covers current knowledge on microtubule-associated tumor suppressors (MATS) in breast cancer and their potential value as prognostic biomarkers. We present recent studies showing that combinatorial expression of ATIP3 and EB1, two microtubule-associated biomarkers with tumor suppressor and oncogenic effects, respectively, improves breast cancer prognosis compared to each biomarker alone. CONCLUSIONS: These findings are discussed regarding the increasing complexity of protein networks composed of MAPs that coordinate microtubule dynamics and functions. Further studies are warranted to evaluate the prognostic value of combined expression of different MATS and their interacting partners in breast cancer.

Regulation of mitotic spindle orientation by phosphorylation of end binding protein 1.

End binding protein 1 (EB1) is a key regulator of microtubule dynamics that orchestrates hierarchical interaction networks at microtubule plus ends to control proper cell division. EB1 activity is known to be regulated by serine/threonine phosphorylation; however, how tyrosine phosphorylation affects EB1 activity remains poorly understood. In this study, we mapped the tyrosine phosphorylation pattern of EB1 in synchronized cells and identified two tyrosine phosphorylation sites (Y217 and Y247) in mitotic cells. Using phospho-deficient (Y/F) and phospho-mimic (Y/D) mutants, we revealed that Y247, but not Y217, is critical for astral microtubule stability. The Y247D mutant contributed to increased spindle angle, indicative of defects in spindle orientation. Time-lapse microscopy revealed that the Y247D mutant significantly delayed mitotic progression by increasing the duration times of prometaphase and metaphase. Structural analysis suggests that Y247 mutants lead to instability of the hydrophobic cavity in the EB homology (EBH) domain, thereby affecting its interactions with p150glued, a protein essential for Gαi/LGN/NuMA complex capture. These findings uncover a crucial role for EB1 phosphorylation in the regulation of mitotic spindle orientation and cell division.

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation.

Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability. Here we have quantitatively characterized the properties of cap size fluctuations during steady-state growth and have developed a theory predicting their timescale and amplitude from the kinetics of microtubule growth and cap maturation. In contrast to growth speed fluctuations, cap size fluctuations show a characteristic timescale, which is defined by the lifetime of the cap sites. Growth fluctuations affect the amplitude of cap size fluctuations; however, cap size does not affect growth speed, indicating that microtubules are far from instability during most of their time of growth. Our theory provides the basis for a quantitative understanding of microtubule stability fluctuations during steady-state growth.

The distinct role of STIM1 and STIM2 in the regulation of store-operated Ca2+ entry and cellular function.

Stromal interaction molecules STIM1 and STIM2 are endoplasmic reticulum (ER) Ca2+ sensors that initiate store-operated Ca 2+ entry (SOCE). The roles of STIM1-mediated SOCE in cancer biology have been highlighted in different types of cancer, but that of STIM2 is unknown. By the model of cervical cancer, here we focus on the cooperative regulation of SOCE by STIM proteins and their distinct roles in cellular function. Immunofluorescent stainings of surgical specimens of cervical cancer show that STIM1 and STIM2 are abundant in tumor tissues, but STIM1 is the major ER Ca 2+ sensor identified in the invasive front of cancer tissues. STIM1 or STIM2 overexpression in cervical cancer SiHa cells induces an upregulated SOCE. Regarding cellular function, STIM1 and STIM2 are necessary for cell proliferation, whereas STIM1 is the dominant ER Ca 2+ sensor involved in cell migration. During SOCE, STIM1 is aggregated and translocated towards the Orai1-containing plasma membrane in association with the microtubule plus-end binding protein EB1. In contrast, STIM2 is constitutively aggregated without significant trafficking or association with microtubules. These results show the distinct role of STIM1 and STIM2 in SOCE and cellular function of cervical cancer cells.

Role of Microtubules and Microtubule-Associated Proteins in HIV-1 Infection.

Recent studies show that human immunodeficiency virus type 1 (HIV-1) can utilize microtubules and their associated proteins to complete key postfusion steps during infection. These include associating with both dynein and kinesin motors, as well as proteins, which enhance infection by altering microtubule dynamics during infection. In this article, we will discuss findings on how dynein and kinesin motors, as well as other microtubule-associated proteins, influence HIV-1 trafficking, viral core uncoating, and nuclear import of the viral ribonucleoprotein (RNP).

Microtubules: A Key to Understand and Correct Neuronal Defects in CDKL5 Deficiency Disorder?

CDKL5 deficiency disorder (CDD) is a severe neurodevelopmental encephalopathy caused by mutations in the X-linked CDKL5 gene that encodes a serine/threonine kinase. CDD is characterised by the early onset of seizures and impaired cognitive and motor skills. Loss of CDKL5 in vitro and in vivo affects neuronal morphology at early and late stages of maturation, suggesting a link between CDKL5 and the neuronal cytoskeleton. Recently, various microtubule (MT)-binding proteins have been identified as interactors of CDKL5, indicating that its roles converge on regulating MT functioning. MTs are dynamic structures that are important for neuronal morphology, migration and polarity. The delicate control of MT dynamics is fundamental for proper neuronal functions, as evidenced by the fact that aberrant MT dynamics are involved in various neurological disorders. In this review, we highlight the link between CDKL5 and MTs, discussing how CDKL5 deficiency may lead to deranged neuronal functions through aberrant MT dynamics. Finally, we discuss whether the regulation of MT dynamics through microtubule-targeting agents may represent a novel strategy for future pharmacological approaches in the CDD field.

Microtubule plus-end tracking of end-binding protein 1 (EB1) is regulated by CDK5 regulatory subunit-associated protein 2.

Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SXIP (Ser-X-Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SXIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SXIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/ß-tubulin binding, and microtubule polymerization. The SXIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SXIP motif alone failed to do so. An SXIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SXIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SXIP-containing chimeras. Our results suggest that disruption of SXIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SXIP-containing +TIPs.