The Role of MAPRE2 and Microtubules in Maintaining Normal Ventricular Conduction.

BACKGROUND: Brugada syndrome is associated with loss-of-function SCN5A variants, yet these account for only ≈20% of cases. A recent genome-wide association study identified a novel locus within MAPRE2, which encodes EB2 (microtubule end-binding protein 2), implicating microtubule involvement in Brugada syndrome. METHODS: A mapre2 knockout zebrafish model was generated using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated protein 9) and validated by Western blot. Larval hearts at 5 days post-fertilization were isolated for voltage mapping and immunocytochemistry. Adult fish hearts were used for ECG, patch clamping, and immunocytochemistry. Morpholinos were injected into embryos at 1-cell stage for knockdown experiments. A transgenic zebrafish line with cdh2 tandem fluorescent timer was used to study adherens junctions. Microtubule plus-end tracking and patch clamping were performed in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) with MAPRE2 knockdown and knockout, respectively. RESULTS: Voltage mapping of mapre2 knockout hearts showed a decrease in ventricular maximum upstroke velocity of the action potential and conduction velocity, suggesting loss of cardiac voltage-gated sodium channel function. ECG showed QRS prolongation in adult knockout fish, and patch clamping showed decreased sodium current density in knockout ventricular myocytes and arrhythmias in knockout iPSC-CMs. Confocal imaging showed disorganized adherens junctions and mislocalization of mature Ncad (N-cadherin) with mapre2 loss of function, associated with a decrease of detyrosinated tubulin. MAPRE2 knockdown in iPSC-CMs led to an increase in microtubule growth velocity and distance, indicating changes in microtubule dynamics. Finally, knockdown of ttl encoding tubulin tyrosine ligase in mapre2 knockout larvae rescued tubulin detyrosination and ventricular maximum upstroke velocity of the action potential. CONCLUSIONS: Genetic ablation of mapre2 led to a decrease in voltage-gated sodium channel function, a hallmark of Brugada syndrome, associated with disruption of adherens junctions, decrease of detyrosinated tubulin as a marker of microtubule stability, and changes in microtubule dynamics. Restoration of the detyrosinated tubulin fraction with ttl knockdown led to rescue of voltage-gated sodium channel-related functional parameters in mapre2 knockout hearts. Taken together, our study implicates microtubule dynamics in the modulation of ventricular conduction.

The centriolar satellite protein Cfap53 facilitates formation of the zygotic microtubule organizing center in the zebrafish embryo.

In embryos of most animal species, the zygotic centrosome is assembled by the centriole derived from the sperm cell and pericentriolar proteins present in the oocyte. This zygotic centrosome acts as a microtubule organizing center (MTOC) to assemble the sperm aster and mitotic spindle. As MTOC formation has been studied mainly in adult cells, very little is known about the formation of the zygotic MTOC. Here, we show that zebrafish (Danio rerio) embryos lacking either maternal or paternal Cfap53, a centriolar satellite protein, arrest during the first cell cycle. Although Cfap53 is dispensable for sperm aster function, it aids proper formation of the mitotic spindle. During cell division, Cfap53 colocalizes with γ-tubulin and with other centrosomal and centriolar satellite proteins at the MTOC. Furthermore, we find that γ-tubulin localization at the MTOC is impaired in the absence of Cfap53. Based on these results, we propose a model in which Cfap53 deposited in the oocyte and the sperm participates in the organization of the zygotic MTOC to allow mitotic spindle formation.

Generation of XPA p.Arg228T mutant LUMCi004-A cell line for modeling Xeroderma pigmentosum group A.

Xeroderma pigmentosum group A (XPA) is an inherited skin disorder characterized by sensitivity to ultraviolet radiation. In Maghrebi patients, a homozygous mutation in exon 6 of the XPA gene (c.682C>T) results in the introduction of a premature termination codon. Using CRISPR/Cas9-mediated gene editing, this mutation was introduced into the well-characterized LUMCi004-A line. The resulting hiPSC line showed typical morphology, expressed markers of the undifferentiated state, was able to differentiate into the three germ layers in vitro and displayed a normal karyotype. When paired with its isogenic counterpart, this line represents a valuable resource to model the disease.

Microtubule plus-end tracking proteins SLAIN1/2 and ch-TOG promote axonal development.

Development, polarization, structural integrity, and plasticity of neuronal cells critically depend on the microtubule network and its dynamic properties. SLAIN1 and SLAIN2 are microtubule plus-end tracking proteins that have been recently identified as regulators of microtubule dynamics. SLAINs are targeted to microtubule tips through an interaction with the core components of microtubule plus-end tracking protein network, End Binding family members. SLAINs promote persistent microtubule growth by recruiting the microtubule polymerase ch-TOG to microtubule plus-ends. Here, we show that SLAIN1/2 and ch-TOG-proteins are highly enriched in brain and are expressed throughout mouse brain development. Disruption of the SLAIN-ch-TOG complex in cultured primary rat hippocampal neurons by RNA interference-mediated knockdown and a dominant-negative approach perturbs microtubule growth by increasing catastrophe frequency and inhibits axon extension during neuronal development. Our study shows that proper control of microtubule dynamics is important for axon elongation in developing neurons.

Generation of AAVS1 and CLYBL STRAIGHT-IN v2 acceptor human iPSC lines for integrating DNA payloads.

STRAIGHT-IN is a platform to precisely integrate DNA payloads into the genome of cells, including hiPSCs. Here, we generated two hiPSC acceptor lines each with one copy of an upgraded landing pad (LP). This improved design allows more efficient (∼100 %) and rapid (∼2-3 weeks) generation of genetically modified hiPSC lines containing the desired payloads. This new LP version was inserted into either the AAVS1 (LUMCi004-A-1) or CLYBL (LUMCi004-A-2) safe harbour loci in the hiPSC line, LUMC0099iCTRL04. The resulting lines can be used for the targeted integration of a wide range of transgenes, thereby making them suitable for numerous research applications.

STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER.

Stromal interaction molecule 1 (STIM1) is a transmembrane protein that is essential for store-operated Ca(2+) entry, a process of extracellular Ca(2+) influx in response to the depletion of Ca(2+) stores in the endoplasmic reticulum (ER) (reviewed in [1-4]). STIM1 localizes predominantly to the ER; upon Ca(2+) release from the ER, STIM1 translocates to the ER-plasma membrane junctions and activates Ca(2+) channels (reviewed in [1-4]). Here, we show that STIM1 directly binds to the microtubule-plus-end-tracking protein EB1 and forms EB1-dependent comet-like accumulations at the sites where polymerizing microtubule ends come in contact with the ER network. Therefore, the previously observed tubulovesicular motility of GFP-STIM1 [5] is not a motor-based movement but a traveling wave of diffusion-dependent STIM1 concentration in the ER membrane. STIM1 overexpression strongly stimulates ER extension occurring through the microtubule "tip attachment complex" (TAC) mechanism [6, 7], a process whereby an ER tubule attaches to and elongates together with the EB1-positive end of a growing microtubule. Depletion of STIM1 and EB1 decreases TAC-dependent ER protrusion, indicating that microtubule growth-dependent concentration of STIM1 in the ER membrane plays a role in ER remodeling.

Regulation of microtubule dynamic instability.

Proper regulation of MT (microtubule) dynamics is essential for various vital processes, including the segregation of chromosomes, directional cell migration and differentiation. MT assembly and disassembly is modulated by a complex network of intracellular factors that co-operate or antagonize each other, are highly regulated in space and time and are thus attuned to the cell cycle and differentiation processes. While we only begin to appreciate how the concerted action of MT stabilizers and destabilizers shapes different MT patterns, a clear picture of how individual factors affect the MT structure is emerging. In this paper, we review the current knowledge about proteins that modulate MT dynamic instability.

TORC1 signaling exerts spatial control over microtubule dynamics by promoting nuclear export of Stu2.

The target of rapamycin complex 1 (TORC1) is a highly conserved multiprotein complex that functions in many cellular processes, including cell growth and cell cycle progression. In this study, we define a novel role for TORC1 as a critical regulator of nuclear microtubule (MT) dynamics in the budding yeast Saccharomyces cerevisiae This activity requires interactions between EB1 and CLIP-170 plus end-tracking protein (+TIP) family members with the TORC1 subunit Kog1/Raptor, which in turn allow the TORC1 proximal kinase Sch9/S6K1 to regulate the MT polymerase Stu2/XMAP215. Sch9-dependent phosphorylation of Stu2 adjacent to a nuclear export signal prevents nuclear accumulation of Stu2 before cells enter mitosis. Mutants impaired in +TIP-TORC1 interactions or Stu2 nuclear export show increased nuclear but not cytoplasmic MT length and display nuclear fusion, spindle positioning, and elongation kinetics defects. Our results reveal key mechanisms by which TORC1 signaling controls Stu2 localization and thereby contributes to proper MT cytoskeletal organization in interphase and mitosis.

Non-catalytic motor domains enable processive movement and functional diversification of the kinesin-14 Kar3.

Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a 'foothold' that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus.

CFEOM1-associated kinesin KIF21A is a cortical microtubule growth inhibitor.

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/ß1 and the components of the LL5ß-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis.

A Proteome-wide screen for mammalian SxIP motif-containing microtubule plus-end tracking proteins.

Microtubule plus-end tracking proteins (+TIPs) are structurally and functionally diverse factors that accumulate at the growing microtubule plus-ends, connect them to various cellular structures, and control microtubule dynamics [1, 2]. EB1 and its homologs are +TIPs that can autonomously recognize growing microtubule ends and recruit to them a variety of other proteins. Numerous +TIPs bind to end binding (EB) proteins through natively unstructured basic and serine-rich polypeptide regions containing a core SxIP motif (serine-any amino acid-isoleucine-proline) [3]. The SxIP consensus sequence is short, and the surrounding sequences show high variability, raising the possibility that undiscovered SxIP containing +TIPs are encoded in mammalian genomes. Here, we performed a proteome-wide search for mammalian SxIP-containing +TIPs by combining biochemical and bioinformatics approaches. We have identified a set of previously uncharacterized EB partners that have the capacity to accumulate at the growing microtubule ends, including protein kinases, a small GTPase, centriole-, membrane-, and actin-associated proteins. We show that one of the newly identified +TIPs, CEP104, interacts with CP110 and CEP97 at the centriole and is required for ciliogenesis. Our study reveals the complexity of the mammalian +TIP interactome and provides a basis for investigating the molecular crosstalk between microtubule ends and other cellular structures.

A complex of Kif18b and MCAK promotes microtubule depolymerization and is negatively regulated by Aurora kinases.

INTRODUCTION: Spindle assembly requires tight control of microtubule (MT) dynamics. This is dependent on a variety of MT binding proteins and their upstream regulators. The Aurora kinases have several well-described functions during cell division, but it remains unclear whether they control global spindle microtubule dynamics. RESULTS: Here, we find that simultaneous inhibition of Aurora A and B results in a dramatic decrease in spindle MT stability, and we identify the uncharacterized kinesin-8 Kif18b as a mediator of this effect. In interphase, Kif18b is nuclear, but upon nuclear envelope breakdown, Kif18b binds to astral MT plus ends through an interaction with EB1. Surprisingly, Kif18b also binds to the kinesin-13 motor MCAK, and this interaction is required for robust MT depolymerization. Furthermore, the Kif18b-MCAK interaction is negatively regulated by Aurora kinases through phosphorylation of MCAK, indicating that Aurora kinases regulate MT plus-end stability in mitosis through control of Kif18b-MCAK complex formation. CONCLUSION: Together, these results uncover a novel role for Aurora kinases in regulating spindle MT dynamics through Kif18b-MCAK and suggest that the Kif18b-MCAK complex constitutes the major MT plus-end depolymerizing activity in mitotic cells.

SLAIN2 links microtubule plus end-tracking proteins and controls microtubule growth in interphase.

The ends of growing microtubules (MTs) accumulate a set of diverse factors known as MT plus end-tracking proteins (+TIPs), which control microtubule dynamics and organization. In this paper, we identify SLAIN2 as a key component of +TIP interaction networks. We showed that the C-terminal part of SLAIN2 bound to end-binding proteins (EBs), cytoplasmic linker proteins (CLIPs), and CLIP-associated proteins and characterized in detail the interaction of SLAIN2 with EB1 and CLIP-170. Furthermore, we found that the N-terminal part of SLAIN2 interacted with ch-TOG, the mammalian homologue of the MT polymerase XMAP215. Through its multiple interactions, SLAIN2 enhanced ch-TOG accumulation at MT plus ends and, as a consequence, strongly stimulated processive MT polymerization in interphase cells. Depletion or disruption of the SLAIN2-ch-TOG complex led to disorganization of the radial MT array. During mitosis, SLAIN2 became highly phosphorylated, and its interaction with EBs and ch-TOG was inhibited. Our study provides new insights into the molecular mechanisms underlying cell cycle-specific regulation of MT polymerization and the organization of the MT network.

Rac1 and Rac3 have opposing functions in cell adhesion and differentiation of neuronal cells.

Rac1 and Rac3 are highly homologous members of the Rho small GTPase family. Rac1 is ubiquitously expressed and regulates cell adhesion, migration and differentiation in various cell types. Rac3 is primarily expressed in brain and may therefore have a specific function in neuronal cells. We found that depletion of Rac1 by short interference RNA leads to decreased cell-matrix adhesions and cell rounding in neuronal N1E-115 cells. By contrast, depletion of Rac3 induces stronger cell adhesions and dramatically increases the outgrowth of neurite-like protrusions, suggesting opposite functions for Rac1 and Rac3 in neuronal cells. Consistent with this, overexpression of Rac1 induces cell spreading, whereas overexpression of Rac3 results in a contractile round morphology. Rac1 is mainly found at the plasma membrane, whereas Rac3 is predominantly localized in the perinuclear region. Residues 185-187, present in the variable polybasic rich region at the carboxyl terminus are responsible for the difference in phenotype induced by Rac1 and Rac3 as well as for their different intracellular localization. The Rac1-opposing function of Rac3 is not mediated by or dependent on components of the RhoA signaling pathway. It rather seems that Rac3 exerts its function through negatively affecting integrin-mediated cell-matrix adhesions. Together, our data reveal that Rac3 opposes Rac1 in the regulation of cell adhesion and differentiation of neuronal cells.