FMRP Long-Range Transport and Degradation Are Mediated by Dynlrb1 in Sensory Neurons.

The fragile X messenger ribonucleoprotein 1 (FMRP) is a multifunctional RNA-binding protein implicated in human neurodevelopmental and neurodegenerative disorders. FMRP mediates the localization and activity-dependent translation of its associated mRNAs through the formation of phase-separated condensates that are trafficked by microtubule-based motors in axons. Axonal transport and localized mRNA translation are critical processes for long-term neuronal survival and are closely linked to the pathogenesis of neurological diseases. FMRP dynein-mediated axonal trafficking is still largely unexplored but likely to constitute a key process underlying FMRP spatiotemporal translational regulation. Here, we show that dynein light chain roadblock 1 (Dynlrb1), a subunit of the dynein complex, is a critical regulator of FMRP function. In sensory axons, FMRP associates with endolysosomal organelles, likely through annexin A11, and is retrogradely trafficked by the dynein complex in a Dynlrb1-dependent manner. Moreover, Dynlrb1 silencing induced FMRP granule accumulation and repressed the translation of microtubule-associated protein 1b, one of its primary mRNA targets. Our findings suggest that Dynlrb1 regulates FMRP function through the control of its transport and targeted degradation.

Microtubule and Actin Differentially Regulate Synaptic Vesicle Cycling to Maintain High-Frequency Neurotransmission.

Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission.SIGNIFICANCE STATEMENT The presence and functional role of MTs in the presynaptic terminal are controversial. Here, we demonstrate that MTs are present near SVs in calyceal presynaptic terminals and that MT depolymerization specifically prolongs the slow-recovery component of EPSCs from short-term depression. In contrast, F-actin depolymerization specifically prolongs fast-recovery component. Depolymerization of MT or F-actin has no direct effect on SV exocytosis/endocytosis or basal transmission, but significantly impairs the fidelity of high-frequency transmission, suggesting that presynaptic cytoskeletal filaments play essential roles in SV replenishment for the maintenance of high-frequency neurotransmission.

Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies.

Giant presynaptic terminal brain slice preparations have allowed intracellular recording of electrical signals and molecular loading, elucidating cellular and molecular mechanisms underlying neurotransmission and modulation. However, molecular genetic manipulation or optical imaging in these preparations is hampered by factors, such as tissue longevity and background fluorescence. To overcome these difficulties, we developed a giant presynaptic terminal culture preparation, which allows genetic manipulation and enables optical measurements of synaptic vesicle dynamics, simultaneously with presynaptic electrical signal recordings. This giant synapse reconstructed from dissociated mouse brainstem neurons resembles the development of native calyceal giant synapses in several respects. Thus, this novel preparation constitutes a powerful tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development. SIGNIFICANCE STATEMENT: We have developed a novel culture preparation of giant mammalian synapses. These presynaptic terminals make it possible to perform optical imaging simultaneously with presynaptic electrophysiological recording. We demonstrate that this enables one to dissect endocytic and acidification times of synaptic vesicles. In addition, developmental elimination and functional maturation in this cultured preparation provide a useful model for studying presynaptic development. Because this giant synapse preparation allows molecular genetic manipulations, it constitutes a powerful new tool for studying molecular mechanisms of neurotransmission, neuromodulation, and neuronal development.

Role of tropomodulin's leucine rich repeat domain in the formation of neurite-like processes.

Actin dynamics is fundamental for neurite development; monomer depolymerization from pointed ends is rate-limiting in actin treadmilling. Tropomodulins (Tmod) make up a family of actin pointed end-capping proteins. Of the four known isoforms, Tmod1-Tmod3 are expressed in brain cells. We investigated the role of Tmod's C-terminal (LRR) domain in the formation of neurite-like processes by overexpressing Tmod1 and Tmod2 with deleted or mutated LRR domains in PC12 cells, a model system used to study neuritogenesis. Tmod1 overexpression results in a normal quantity and a normal length of processes, while Tmod2 overexpression reduces both measures. The Tmod2 overexpression phenotype is mimicked by overexpression of Tmod1 with the LRR domain removed or with three point mutations in the LRR domain that disrupt exposed clusters of conserved residues. Removal of Tmod2's LRR domain does not significantly alter the outgrowth of neurite-like processes compared to that of Tmod2. Overexpression of chimeras with the N-terminal and C-terminal domains switched between Tmod1 and Tmod2 reinforces the idea that Tmod1's LRR domain counteracts the reductive effect of the Tmod N-terminal domain upon formation of processes while Tmod2's LRR domain does not. We suggest that the TM-dependent actin capping ability of both Tmods inhibits the formation of processes, but in Tmod1, this inhibition can be controlled via its LRR domain. Circular dichroism, limited proteolysis, and molecular dynamics demonstrate structural differences in the C-terminal region of the LRR domains of Tmod1, Tmod2, and the Tmod1 mutant.

Hacd2 deficiency in mice leads to an early and lethal mitochondrial disease.

OBJECTIVE: Mitochondria fuel most animal cells with ATP, ensuring proper energetic metabolism of organs. Early and extensive mitochondrial dysfunction often leads to severe disorders through multiorgan failure. Hacd2 gene encodes an enzyme involved in very long chain fatty acid (C ≥ 18) synthesis, yet its roles in vivo remain poorly understood. Since mitochondria function relies on specific properties of their membranes conferred by a particular phospholipid composition, we investigated if Hacd2 gene participates to mitochondrial integrity. METHODS: We generated two mouse models, the first one leading to a partial knockdown of Hacd2 expression and the second one, to a complete knockout of Hacd2 expression. We performed an in-depth analysis of the associated phenotypes, from whole organism to molecular scale. RESULTS: Thanks to these models, we show that Hacd2 displays an early and broad expression, and that its deficiency in mice is lethal. Specifically, partial knockdown of Hacd2 expression leads to death within one to four weeks after birth, from a sudden growth arrest followed by cachexia and lethargy. The total knockout of Hacd2 is even more severe, characterized by embryonic lethality around E9.5 following developmental arrest and pronounced cardiovascular malformations. In-depth mechanistic analysis revealed that Hacd2 deficiency causes altered mitochondrial efficiency and ultrastructure, as well as accumulation of oxidized cardiolipin. CONCLUSIONS: Altogether, these data indicate that the Hacd2 gene is essential for energetic metabolism during embryonic and postnatal development, acting through the control of proper mitochondrial organization and function.

Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model.

Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer's disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.

Development of 3D culture scaffolds for directional neuronal growth using 2-photon lithography.

Conventional applications of transplant technology, applied to severe traumatic injuries of the nervous system, have met limited success in the clinics due to the complexity of restoring function to the damaged tissue. Neural tissue engineering aims to deploy scaffolds mimicking the physiological properties of the extracellular matrix to facilitate the elongation of axons and the repair of damaged nerves. However, the fabrication of ideal scaffolds with precisely controlled thickness, texture, porosity, alignment, and with the required mechanical strength, features needed for effective clinical applications, remains technically challenging. We took advantage of state-of-the-art 2-photon photolithography to fabricate highly ordered and biocompatible 3D nanogrid structures to enhance neuronal directional growth. First, we characterized the physical and chemical properties and proved the biocompatibility of said scaffolds by successfully culturing primary sensory and motor neurons on their surface. Interestingly, axons extended along the fibers with a high degree of alignment to the pattern of the nanogrid, as opposed to the lack of directionality observed on flat glass or polymeric surfaces, and could grow in 3D between different layers of the scaffold. The axonal growth pattern observed is highly desirable for the treatment of traumatic nerve damage occurring during peripheral and spinal cord injuries. Thus, our findings provide a proof of concept and explore the possibility of deploying aligned fibrous 3D scaffold/implants for the directed growth of axons, and could be used in the design of scaffolds targeted towards the restoration and repair of lost neuronal connections.

Cardiolipin content controls mitochondrial coupling and energetic efficiency in muscle.

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

Anterograde Axonal Transport in Neuronal Homeostasis and Disease.

Neurons are highly polarized cells with an elongated axon that extends far away from the cell body. To maintain their homeostasis, neurons rely extensively on axonal transport of membranous organelles and other molecular complexes. Axonal transport allows for spatio-temporal activation and modulation of numerous molecular cascades, thus playing a central role in the establishment of neuronal polarity, axonal growth and stabilization, and synapses formation. Anterograde and retrograde axonal transport are supported by various molecular motors, such as kinesins and dynein, and a complex microtubule network. In this review article, we will primarily discuss the molecular mechanisms underlying anterograde axonal transport and its role in neuronal development and maturation, including the establishment of functional synaptic connections. We will then provide an overview of the molecular and cellular perturbations that affect axonal transport and are often associated with axonal degeneration. Lastly, we will relate our current understanding of the role of axonal trafficking concerning anterograde trafficking of mRNA and its involvement in the maintenance of the axonal compartment and disease.

Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core.

Nuclear pore complex (NPC) is a gating nanomachine with a central selective barrier composed mainly of Nups, which contain intrinsically disordered (non-structured) regions (IDRs) with phenylalanine-glycine (FG) motifs (FG-NUPs). The NPC central FG network dynamics is poorly understood, as FG-NUPs liquid-liquid phase separation (LLPS) have evaded structural characterization. Moreover, the working mechanism of single FG-NUP-biofilaments residing at the central lumen is unknown. In general, flexible biofilaments are expected to be tangled and knotted during their motion and interaction. However, filament knotting visualization in real-time and space has yet to be visualized at the nanoscale. Here, we report a spatiotemporally tracking method for FG-NUP organization with nanoscale resolution, unveiling FG-NUP conformation in NPCs of colorectal cells and organoids at timescales of ~150 ms using high-speed atomic force microscopy (HS-AFM). Tracking of FG-NUP single filaments revealed that single filaments have a heterogeneous thickness in normal and cancer models which in turn affected the filament rotation and motion. Notably, FG-NUPs are overexpressed in various cancers. Using the FG-NUP inhibitor, trans-1,2-cyclohexanediol, we found that central plug size was significantly reduced and incompletely reversible back to filamentous structures in aggressive colon cancer cells and organoids. These data showed a model of FG-NUPs reversible self-assembly devolving into the central plug partial biogenesis. Taken together, HS-AFM enabled the tracking and manipulation of single filaments of native FG-NUPs which has remained evasive for decades.

Culture of Mouse Giant Central Nervous System Synapses and Application for Imaging and Electrophysiological Analyses.

Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.

The role of epidermal growth factor and its receptors in mammalian CNS.

Epidermal growth factor (EGF) is a common mitogenic factor that stimulates the proliferation of different types of cells, especially fibroblasts and epithelial cells. EGF activates the EGF receptor (EGFR/ErbB), which initiates, in turn, intracellular signaling. EGFR family is also expressed in neurons of the hippocampus, cerebellum, and cerebral cortex in addition to other regions of the central nervous system (CNS). EGF enhances the differentiation, maturation and survival of a variety of neurons. Transgenic mice lacking the EGFR developed neurodegenerative disease and die within the first month of birth. EGF acts not only on mitotic cells but also on postmitotic neurons, and many studies have indicated that EGF has neuromodulatory effect on various types of neurons in the CNS. This review highlights some of the major recent findings pertinent to the EGF and ErbB family with special references to elucidating their roles in the regulation of neurogenesis, signal transduction and trafficking and degradation.

Presynaptic morphology and vesicular composition determine vesicle dynamics in mouse central synapses.

Transport of synaptic vesicles (SVs) in nerve terminals is thought to play essential roles in maintenance of neurotransmission. To identify factors modulating SV movements, we performed real-time imaging analysis of fluorescently labeled SVs in giant calyceal and conventional hippocampal terminals. Compared with small hippocampal terminals, SV movements in giant calyceal terminals were faster, longer and kinetically more heterogeneous. Morphological maturation of giant calyceal terminals was associated with an overall reduction in SV mobility and displacement heterogeneity. At the molecular level, SVs over-expressing vesicular glutamate transporter 1 (VGLUT1) showed higher mobility than VGLUT2-expressing SVs. Pharmacological disruption of the presynaptic microtubule network preferentially reduced long directional movements of SVs between release sites. Functionally, synaptic stimulation appeared to recruit SVs to active zones without significantly altering their mobility. Hence, the morphological features of nerve terminals and the molecular signature of vesicles are key elements determining vesicular dynamics and movements in central synapses.

KIF17 dynamics and regulation of NR2B trafficking in hippocampal neurons.

KIF17, a recently characterized member of the kinesin superfamily proteins, has been proposed to bind in vitro to a protein complex containing mLin10 (Mint1/X11) and the NR2B subunit of the NMDA receptors (NMDARs). In the mammalian brain, NMDARs play an important role in synaptic plasticity, learning, and memory. Here we present, for the first time, the dynamic properties of KIF17 and provide evidence of its function in the transport of NR2B in living mammalian neurons. KIF17 vesicles enter and move specifically along dendrites in a processive way, at an average speed of 0.76 microm/sec. These vesicles are effectively associated with extrasynaptic NR2B, and thus they transport and deliver NR2B subunits in dendrites. However, KIF17 does not seem to enter directly into postsynaptic regions. Cellular knockdown or functional blockade of KIF17 significantly impairs NR2B expression and its synaptic localization. Interestingly, the decrease in the number of synaptic NR2B subunits is followed by a parallel increase in the number of NR2A subunits at synapses. In contrast, upregulation of the expression level of NR2B, after treatment with the NMDAR antagonist D(-)-2-amino-5-phosphonopentanoic acid, simultaneously increases the expression level of KIF17. These observations concerning the downregulation or upregulation of KIF17 and NR2B reveal the probable existence of a shared regulation process between the motor and its cargo. Taken together, these results illustrate the complex mechanisms underlying the active transport and regulation of NR2B by the molecular motor KIF17 in living hippocampal neurons.

HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.

The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism.

Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes.

Deafness is a common sensory defect in human. Our understanding of the molecular bases of this pathology comes from the study of a few genes that have been identified in human and/or in mice. Indeed, deaf mouse mutants are good models for studying and identifying genes involved in human hereditary hearing loss. Among these mouse mutants, twister was initially reported to have abnormal behavior and thereafter to be deaf. The recessive twister (twt) mutation has been mapped on mouse Chromosome (Chr) 7, homologous to the long arm of human Chr 15 (15q11). Otog, the gene encoding otogelin, a glycoprotein specific to all the acellular membranes of the inner ear, is also localized to mouse Chr 7, but in a region more proximal to the twister mutation, homologous to the short arm of human Chr 11 (11p15) carrying the two deafness loci, DFNB18 and USH1C. Mutant mice resulting from the knock-out of Otog, the Otog(tm1Prs) mice, present deafness and severe imbalance. Although twt had been mapped distally to Otog, these data prompted us to test whether twt could be due to a mutation in the Otog locus. Here, we demonstrate by genetic analysis that twt is actually allelic to Otog(tm1Prs). We further extend the phenotypical analysis of twister mice, documenting the association of a severe vestibular phenotype and moderate to severe form of deafness. Molecular analysis of the Otog gene revealed the absence of detectable expression of Otog in the twister mutant. The molecular and phenotypical description of the twt mouse mutation, Otog(twt), reported herein, highlights the importance of the acellular membranes in the inner ear mechanotransduction process.

Mutations changing tropomodulin affinity for tropomyosin alter neurite formation and extension.

Assembly of the actin cytoskeleton is an important part of formation of neurites in developing neurons. Tropomodulin, a tropomyosin-dependent capping protein for the pointed end of the actin filament, is one of the key players in this process. Tropomodulin binds tropomyosin in two binding sites. Tmod1 and Tmod2, tropomodulin isoforms found in neurons, were overexpressed in PC12 cells, a model system for neuronal differentiation. Tmod1 did not affect neuronal differentiation; while cells expressing Tmod2 showed a significant reduction in the number and the length of neurites. Both tropomodulins bind short α-, γ- and δ-tropomyosin isoforms. Mutations in one of the tropomyosin-binding sites of Tmod1, which increased its affinity to short γ- and δ-tropomyosin isoforms, caused a decrease in binding short α-tropomyosin isoforms along with a 2-fold decrease in the length of neurites. Our data demonstrate that Tmod1 is involved in neuronal differentiation for proper neurite formation and outgrowth, and that Tmod2 inhibits these processes. The mutations in the tropomyosin-binding site of Tmod1 impair neurite outgrowth, suggesting that the integrity of this binding site is critical for the proper function of Tmod1 during neuronal differentiation.

A molecular motor, KIF13A, controls anxiety by transporting the serotonin type 1A receptor.

Molecular motors are fundamental to neuronal morphogenesis and function. However, the extent to which molecular motors are involved in higher brain functions remains largely unknown. In this study, we show that mice deficient in the kinesin family motor protein KIF13A (Kif13a(-/-) mice) exhibit elevated anxiety-related behavioral phenotypes, probably because of a reduction in 5HT(1A) receptor (5HT(1A)R) transport. The cell-surface expression level of the 5HT(1A)R was reduced in KIF13A-knockdown neuroblastoma cells and Kif13a(-/-) hippocampal neurons. Biochemical analysis showed that the forkhead-associated (FHA) domain of KIF13A and an intracellular loop of the 5HT(1A)R are the interface between the motor and cargo vesicles. A minimotor consisting of the motor and FHA domains is able to transport 5HT(1A)R-carrying organelles in in vitro reconstitution assays. Collectively, our results suggest a role for this molecular motor in anxiety control.

Pathology influences blood pressure change following vagal stimulation in an animal intubation model.

PURPOSE: The haemodynamic response to critical care intubation is influenced by the use of sedation and relaxant drugs and the activation of the vagal reflex. It has been hypothesized that different disease states may have a contrasting effect on the cardiovascular response to vagal stimulation. Our objective was to determine whether the blood pressure response to vagal stimulation was modified by endotoxaemia or hypovolaemia. METHODS: New Zealand White rabbits were anaesthetised with urethane before tracheotomy. The exposed left Vagus nerve of randomised groups of control (n = 11), endotoxin (n = 11, 1 mg/kg), hypovolaemia 40% (n = 8) and hypovolaemia 20% (n = 8) rabbits were subjected to 10 Hz pulsed electrical stimulations of 25 s duration every 15 min. Haemodynamic parameters were recorded from a catheter in the right carotid artery connected to an iWorx monitor. Serum catecholamines were measured every 30 min using reverse-phase ion-pairing liquid chromatography. The change in blood pressure after vagal stimulation was compared to controls for one hour after the first death in the experimental groups. RESULTS: 29% of the rabbits died in the hypovolaemia 40% group and 27% in the endotoxin group. One rabbit died in the hypovolaemia 40% group before vagal stimulation and was excluded. Following electrical stimulation of the Vagus nerve there was a fall in blood pressure in control rabbits. Blood pressure was conserved in the hypovolaemic rabbits compared to controls (p<0.01). For the endotoxaemic rabbits, there was a non-significant trend for the mean blood pressure to decrease more than the controls. Serum catecholamines were significantly raised in both the hypovolaemic and endotoxaemic rabbits. CONCLUSIONS: Pathology may contribute to modifications in blood pressure when vagal activation occurs. Patients who are either already vasoconstricted, or not vasoplegic, may be less at risk from intubation-related vagally mediated reductions in blood pressure than those with vasodilatory pathologies.

Bmcc1s, a novel brain-isoform of Bmcc1, affects cell morphology by regulating MAP6/STOP functions.

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology.