CLASP2 safeguards hematopoietic stem cell properties during mouse and fish development.

Hematopoietic stem cells (HSCs) express a large variety of cell surface receptors that are associated with acquisition of self-renewal and multipotent properties. Correct expression of these receptors depends on a delicate balance between cell surface trafficking, recycling, and degradation and is controlled by the microtubule network and Golgi apparatus, whose roles have hardly been explored during embryonic/fetal hematopoiesis. Here we show that, in the absence of CLASP2, a microtubule-associated protein, the overall production of HSCs is reduced, and the produced HSCs fail to self-renew and maintain their stemness throughout mouse and zebrafish development. This phenotype can be attributed to decreased cell surface expression of the hematopoietic receptor c-Kit, which originates from increased lysosomal degradation in combination with a reduction in trafficking to the plasma membrane. A dysfunctional Golgi apparatus in CLASP2-deficient HSCs seems to be the underlying cause of the c-Kit expression and signaling imbalance.

CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells.

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

Novel TUBA4A Variant Associated With Familial Frontotemporal Dementia.

OBJECTIVE: Despite the strong genetic component of frontotemporal dementia (FTD), a substantial proportion of patients remain genetically unresolved. We performed an in-depth study of a family with an autosomal dominant form of FTD to investigate the underlying genetic cause. METHODS: Following clinical and pathologic characterization of the family, genetic studies included haplotype sharing analysis and exome sequencing. Subsequently, we performed immunohistochemistry, immunoblotting, and a microtubule repolymerization assay to investigate the potential impact of the candidate variant in tubulin alpha 4a (TUBA4A). RESULTS: The clinical presentation in this family is heterogeneous, including behavioral changes, parkinsonian features, and uncharacterized dementia. Neuropathologic examination of 2 patients revealed TAR DNA binding protein 43 (TDP-43) pathology with abundant dystrophic neurites and neuronal intranuclear inclusions, consistent with frontotemporal lobar degeneration-TDP type A. We identified a likely pathogenic variant in TUBA4A segregating with disease. TUBA4A encodes for α-tubulin, which is a major component of the microtubule network. Variants in TUBA4A have been suggested as a rare genetic cause of amyotrophic lateral sclerosis (ALS) and have sporadically been reported in patients with FTD without supporting genetic segregation. A decreased trend of TUBA4A protein abundance was observed in patients compared with controls, and a microtubule repolymerization assay demonstrated disrupted α-tubulin function. As opposed to variants found in ALS, TUBA4A variants associated with FTD appear more localized to the N-terminus, indicating different pathogenic mechanisms. CONCLUSIONS: Our findings support the role of TUBA4A variants as rare genetic cause of familial FTD.

Distinct Functions for Mammalian CLASP1 and -2 During Neurite and Axon Elongation.

Mammalian cytoplasmic linker associated protein 1 and -2 (CLASP1 and -2) are microtubule (MT) plus-end tracking proteins that selectively stabilize MTs at the edge of cells and that promote MT nucleation and growth at the Golgi, thereby sustaining cell polarity. In vitro analysis has shown that CLASPs are MT growth promoting factors. To date, a single CLASP1 isoform (called CLASP1α) has been described, whereas three CLASP2 isoforms are known (CLASP2α, -ß, and -γ). Although CLASP2ß/γ are enriched in neurons, suggesting isoform-specific functions, it has been proposed that during neurite outgrowth CLASP1 and -2 act in a redundant fashion by modulating MT dynamics downstream of glycogen synthase kinase 3 (GSK3). Here, we show that in differentiating N1E-115 neuroblastoma cells CLASP1 and CLASP2 differ in their accumulation at MT plus-ends and display different sensitivity to GSK3-mediated phosphorylation, and hence regulation. More specifically, western blot (WB) analysis suggests that pharmacological inhibition of GSK3 affects CLASP2 but not CLASP1 phosphorylation and fluorescence-based microscopy data show that GSK3 inhibition leads to an increase in the number of CLASP2-decorated MT ends, as well as to increased CLASP2 staining of individual MT ends, whereas a reduction in the number of CLASP1-decorated ends is observed. Thus, in N1E-115 cells CLASP2 appears to be a prominent target of GSK3 while CLASP1 is less sensitive. Surprisingly, knockdown of either CLASP causes phosphorylation of GSK3, pointing to the existence of feedback loops between CLASPs and GSK3. In addition, CLASP2 depletion also leads to the activation of protein kinase C (PKC). We found that these differences correlate with opposite functions of CLASP1 and CLASP2 during neuronal differentiation, i.e., CLASP1 stimulates neurite extension, whereas CLASP2 inhibits it. Consistent with knockdown results in N1E-115 cells, primary Clasp2 knockout (KO) neurons exhibit early accelerated neurite and axon outgrowth, showing longer axons than control neurons. We propose a model in which neurite outgrowth is fine-tuned by differentially posttranslationally modified isoforms of CLASPs acting at distinct intracellular locations, thereby targeting MT stabilizing activities of the CLASPs and controlling feedback signaling towards upstream kinases. In summary, our findings provide new insight into the roles of neuronal CLASPs, which emerge as regulators acting in different signaling pathways and locally modulating MT behavior during neurite/axon outgrowth.

A Cell Culture System to Investigate the Presynaptic Control of Subsynaptic Membrane Differentiation at the Neuromuscular Junction.

For decades the neuromuscular junction (NMJ) has been a favorite preparation to investigate basic mechanisms of synaptic function and development. As its function is to transmit action potentials in a 1:1 ratio from motor neurons to muscle fibers, the NMJ shows little or no functional plasticity, a property that makes it poorly suited to investigate mechanisms of use-dependent adaptations of synaptic function, which are thought to underlie learning and memory formation in the brain. On the other hand, the NMJ is unique in that the differentiation of the subsynaptic membrane is regulated by one major factor secreted from motor neurons, agrin. As a consequence, myotubes grown on a laminin substrate that is focally impregnated with recombinant neural agrin closely resemble the situation in vivo, where agrin secreted from motor neurons binds to the basal lamina of the NMJ's synaptic cleft to induce and maintain the subsynaptic muscle membrane. We provide here a detailed protocol through which acetylcholine receptor clusters are induced in cultured myotubes contacting laminin-attached agrin, enabling molecular, biochemical and cell biological analyses including high resolution microscopy in 4D. This preparation is ideally suited to investigate the mechanisms involved in the assembly of the postsynaptic muscle membrane, providing distinct advantages over inducing AChR clusters using soluble agrin.

Isolation of Functional Tubulin Dimers and of Tubulin-Associated Proteins from Mammalian Cells.

The microtubule (MT) cytoskeleton forms a dynamic filamentous network that is essential for many processes, including mitosis, cell polarity and shape, neurite outgrowth and migration, and ciliogenesis [1, 2]. MTs are built up of α/ß-tubulin heterodimers, and their dynamic behavior is in part regulated by tubulin-associated proteins (TAPs). Here we describe a novel system to study mammalian tubulins and TAPs. We co-expressed equimolar amounts of triple-tagged α-tubulin and ß-tubulin using a 2A "self-cleaving" peptide and isolated functional fluorescent tubulin dimers from transfected HEK293T cells with a rapid two-step approach. We also produced two mutant tubulins that cause brain malformations in tubulinopathy patients [3]. We then applied a paired mass-spectrometry-based method to identify tubulin-binding proteins in HEK293T cells and describe both novel and known TAPs. We find that CKAP5 and the CLASPs, which are MT plus-end-tracking proteins with TOG(L)-domains [4], bind tubulin efficiently, as does the Golgi-associated protein GCC185, which interacts with the CLASPs [5]. The N-terminal TOGL domain of CLASP1 contributes to tubulin binding and allows CLASP1 to function as an autonomous MT-growth-promoting factor. Interestingly, mutant tubulins bind less well to a number of TAPs, including CLASPs and GCC185, and incorporate less efficiently into cellular MTs. Moreover, expression of these mutants in cells impairs several MT-growth-related processes involving TAPs. Thus, stable tubulin-TAP interactions regulate MT nucleation and growth in cells. Combined, our results provide a resource for investigating tubulin interactions and functions and widen the spectrum of tubulin-related disease mechanisms.

CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5ß and actin for focal delivery of acetylcholine receptor vesicles.

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end-tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3ß. Here we show that knockdown of the CLASP2-interaction partner LL5ß by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5ß interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5ß at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3ß, CLASP2, and LL5ß, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.

Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3ß (GSK3ß)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3ß-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3ß-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.

Dynamic microtubules catalyze formation of navigator-TRIO complexes to regulate neurite extension.

Neurite extension is regulated by multiple signaling cascades that ultimately converge on the actin and microtubule networks [1]. Rho GTPases, molecular switches that oscillate between an inactive, GDP-bound state and an active, GTP-bound state, play a pivotal role in controlling actin cytoskeleton dynamics in the growth cone, whereas the dynamic behavior and interactions of microtubules are largely regulated by proteins called plus-end-tracking proteins (+TIPs), which associate with the ends of growing microtubules. Here, we show that the +TIP Navigator 1 (NAV1) is important for neurite outgrowth and interacts and colocalizes with TRIO, a Rho guanine nucleotide exchange factor that enables neurite outgrowth by activating the Rho GTPases Rac1 and RhoG. We find that binding of NAV1 enhances the affinity of TRIO for Rac1 and RhoG, and that NAV1 regulates TRIO-mediated Rac1 activation and neurite outgrowth. TRIO is also a +TIP, as it interacts with the core +TIP EB1 and tracks microtubule plus ends via EB1 and NAV1. Strikingly, the EB1-mediated recruitment of TRIO to microtubule ends is required for proper neurite outgrowth, and stabilization of the microtubule network by paclitaxel affects both the TRIO-NAV1 interaction and the accumulation of these proteins in neurite extensions. We propose that EB1-labeled ends of dynamic microtubules facilitate the formation and localization of functional NAV1-TRIO complexes, which in turn regulate neurite outgrowth by selectively activating Rac1. Our data reveal a novel link between dynamic microtubules, actin cytoskeleton remodeling, and neurite extension.

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane.

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3ß, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

A genetic screen for anchorage-independent proliferation in mammalian cells identifies a membrane-bound neuregulin.

Anchorage-independent proliferation is a hallmark of oncogenic transformation and is thought to be conducive to proliferation of cancer cells away from their site of origin. We have previously reported that primary Schwann cells expressing the SV40 Large T antigen (LT) are not fully transformed in that they maintain a strict requirement for attachment, requiring a further genetic change, such as oncogenic Ras, to gain anchorage-independence. Using the LT-expressing cells, we performed a genetic screen for anchorage-independent proliferation and identified Sensory and Motor Neuron Derived Factor (SMDF), a transmembrane class III isoform of Neuregulin 1. In contrast to oncogenic Ras, SMDF induced enhanced proliferation in normal primary Schwann cells but did not trigger cellular senescence. In cooperation with LT, SMDF drove anchorage-independent proliferation, loss of contact inhibition and tumourigenicity. This transforming ability was shared with membrane-bound class III but not secreted class I isoforms of Neuregulin, indicating a distinct mechanism of action. Importantly, we show that despite being membrane-bound signalling molecules, class III neuregulins transform via a cell intrinsic mechanism, as a result of constitutive, elevated levels of ErbB signalling at high cell density and in anchorage-free conditions. This novel transforming mechanism may provide new targets for cancer therapy.