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Tubulin acetylation in Camsaps-mutated cells. (A) Western blot analysis of acetylated tubulin in N2a cells treated with siRNA specific for CAMSAP3 (siC3) or CAMSAP2 (siC2), or with control siRNA (siCtrl). The ratio of acetylated tubulin (Ace-tub) to total α-tubulin (Tub) was measured by Fiji software, using four independent samples, and the values were normalized by setting the ratio in siCtrl samples to "one." *P < 0.05 versus siCtrl. n.s., not significant. (B and C) Immunostaining for acetylated tubulin (green), MAP2 (blue), and α-tubulin (red) in neurons at DIV5, derived from wild-type and C3 dc/dc hippocampi. Immunostaining signals were scanned along the dotted lines in C. (D) Immunostaining for acetylated tubulin (red), α-tubulin (white), and DNA (blue) in neurons at DIV6, derived from wild-type, C2 −/− , and C3 dc/dc hippocampi. Cells were treated with 10 μM nocodazole for 1 h at 37 °C before fixation. Arrowheads indicate examples of elongated processes. A representative image from more than 50 neurons obtained from three independent experiments is shown. (E) Western blot analysis of acetylated tubulin in neurons at DIV6, derived from wild-type (wt) and C2 −/− or C3 dc/dc hippocampi. Some cultures were treated with 10 μM nocodazole (NDZ) for 1 h at 37 °C before sample preparation. The ratio of acetylated tubulin (Ace-tub) to total α-tubulin (Tub) was measured by Fiji software, using three independent samples, and the values were normalized by setting the ratio in wt samples to "one." *P < 0.05 versus wt. n.s., not significant. (F) Localization of CAMSAP2 or CAMSAP3 (red), in relation to acetylated (Ace-tub) or tyrosinated (Tyr-tub) tubulins (green) in wild-type neurons at DIV4. Immunostaining signals were scanned along the dotted lines. (Scale bars, 20 μm in B and D; 10 μm in E.)
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Each neuron forms a single axon and multiple dendrites, and this configuration is important for wiring the brain. How only a single axon extends from a neuron, however, remains unknown. This study demonstrates that CAMSAP3, a protein that binds the minus-end of microtubules, preferentially localizes along axons in hippocampal neurons....
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... (Fig. 2 A-C), consistent with a previous report (27). In contrast, CAMSAP3 dc/dc neurons showed polarity defects: Around half of them generated multiple axons, ranging from two to four, as assessed by immunostaining for axon markers ( Fig. 2 A and C). Neurons derived from Camsap3 −/− mice also showed enhanced multiaxon formation (SI Appendix, Fig. S3A). We also depleted (knocked down, KD) CAMSAP3 in wild-type neurons, using specific siRNAs (SI Ap- pendix, Fig. S3B), finding similar defects in neurite differentiation (SI Appendix, Fig. S3C). Thus, various types of CAMSAP3 de- ficiency equally induced multiple axon formation. Immunostaining of CAMSAP3 dc/dc neurons for the mutated ...
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... Around half of them generated multiple axons, ranging from two to four, as assessed by immunostaining for axon markers ( Fig. 2 A and C). Neurons derived from Camsap3 −/− mice also showed enhanced multiaxon formation (SI Appendix, Fig. S3A). We also depleted (knocked down, KD) CAMSAP3 in wild-type neurons, using specific siRNAs (SI Ap- pendix, Fig. S3B), finding similar defects in neurite differentiation (SI Appendix, Fig. S3C). Thus, various types of CAMSAP3 de- ficiency equally induced multiple axon formation. Immunostaining of CAMSAP3 dc/dc neurons for the mutated CAMSAP3 protein detected diffuse signals along neurites, which were never enriched in axons (SI Appendix, Fig. S3D), ...
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... assessed by immunostaining for axon markers ( Fig. 2 A and C). Neurons derived from Camsap3 −/− mice also showed enhanced multiaxon formation (SI Appendix, Fig. S3A). We also depleted (knocked down, KD) CAMSAP3 in wild-type neurons, using specific siRNAs (SI Ap- pendix, Fig. S3B), finding similar defects in neurite differentiation (SI Appendix, Fig. S3C). Thus, various types of CAMSAP3 de- ficiency equally induced multiple axon formation. Immunostaining of CAMSAP3 dc/dc neurons for the mutated CAMSAP3 protein detected diffuse signals along neurites, which were never enriched in axons (SI Appendix, Fig. S3D), indicating that CAMSAP3 binding to microtubule minus-ends is required for it ...
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... Ap- pendix, Fig. S3B), finding similar defects in neurite differentiation (SI Appendix, Fig. S3C). Thus, various types of CAMSAP3 de- ficiency equally induced multiple axon formation. Immunostaining of CAMSAP3 dc/dc neurons for the mutated CAMSAP3 protein detected diffuse signals along neurites, which were never enriched in axons (SI Appendix, Fig. S3D), indicating that CAMSAP3 binding to microtubule minus-ends is required for it to concen- trate in ...
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... formed axons might have acquired a hybrid axon/dendrite nature, unlike authentic axons. When Camsap3 dc/dc neurons were cultured up to maturation, synaptic proteins came to accumulate along neurites, suggesting that axon and dendrite differentiation took place normally in these neurons, despite the excess number of putative axons (SI Appendix, Fig. ...
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... line (32) as a neuronal model, which allowed us to conduct efficient preliminary analy- sis. N2a cells expressed both CAMSAP2 and CAMSAP3, exhibiting their scattered distribution in the cytoplasm with the highest concentration at perinuclear zones (SI Appendix, Fig. S4A). When these CAMSAPs were knocked down using siRNAs (SI Appendix, Fig. S3B), acetylated tubulin was increased after CAMSAP3 KD, but it did not change or was slightly decreased in CAMSAP2 KD cells (Fig. 3A). Morphologically, N2a cells exhibited short filopodium-like processes at the peripheries, and this shape of cells was not affected by CAMSAP2 KD. In con- trast, ∼30% of CAMSAP3 KD cells produced long ...
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... CAMSAP2 and CAMSAP3, exhibiting their scattered distribution in the cytoplasm with the highest concentration at perinuclear zones (SI Appendix, Fig. S4A). When these CAMSAPs were knocked down using siRNAs (SI Appendix, Fig. S3B), acetylated tubulin was increased after CAMSAP3 KD, but it did not change or was slightly decreased in CAMSAP2 KD cells (Fig. 3A). Morphologically, N2a cells exhibited short filopodium-like processes at the peripheries, and this shape of cells was not affected by CAMSAP2 KD. In con- trast, ∼30% of CAMSAP3 KD cells produced long neurite-like processes, the number of which varied from cell to cell (SI Ap- pendix, Fig. S4B), as observed in N2a cells whose ...
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... neurite-like process for- mation in N2a cells. Next, we observed tubulin acetylation in hippocampal neurons. Immunostaining showed that, in wild-type neurons, acetylation was broadly detected in various regions of the neuron. Never- theless, the ratio of acetylated tubulin to the total tubulin tended to be higher in axons compared with dendrites (Fig. 3B, Upper and C), consistent with previous reports (6,34). In Camsap3 dc/dc neurons, this relative increase of acetylated tubulins came to be observed in multiple neurites (Fig. 3B, Lower and C). When these neurons were treated with nocodazole, nocodazole-resistant, acetylated microtubules were detectable in various regions of a neuron, ...
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... detected in various regions of the neuron. Never- theless, the ratio of acetylated tubulin to the total tubulin tended to be higher in axons compared with dendrites (Fig. 3B, Upper and C), consistent with previous reports (6,34). In Camsap3 dc/dc neurons, this relative increase of acetylated tubulins came to be observed in multiple neurites (Fig. 3B, Lower and C). When these neurons were treated with nocodazole, nocodazole-resistant, acetylated microtubules were detectable in various regions of a neuron, regardless of whether CAMSAPs were present or not. However, concerning the thin and long processes extending from the cell body, which are putative axons, only single processes ...
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... of a neuron, regardless of whether CAMSAPs were present or not. However, concerning the thin and long processes extending from the cell body, which are putative axons, only single processes held nocodazole-resistant microtubules in most of CAMSAP2 knock- out or wild-type neurons, whereas multiple processes had them in CAMSAP3-mutated neurons (Fig. 3D). These observations sup- port the idea that CAMSAP3 dysfunction leads to an increase of neurites with stable microtubules also in ...
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... in N2a cells, however, in hippocampal neurons, the total level of acetylated tubulin did not particularly change in Camsap2 or Camsap3 mutants, as assessed by Western blotting (Fig. 3E). This was also the case for cells treated with nocoda- zole. Immunostaining for acetylated tubulin also did not suggest apparent differences in its overall level between these samples (Fig. 3 B and D). To investigate whether CAMSAP3 muta- tions could affect tubulin dynamics in hippocampal neurons, we closely observed the distribution ...
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... in hippocampal neurons, the total level of acetylated tubulin did not particularly change in Camsap2 or Camsap3 mutants, as assessed by Western blotting (Fig. 3E). This was also the case for cells treated with nocoda- zole. Immunostaining for acetylated tubulin also did not suggest apparent differences in its overall level between these samples (Fig. 3 B and D). To investigate whether CAMSAP3 muta- tions could affect tubulin dynamics in hippocampal neurons, we closely observed the distribution of CAMSAPs in relation to that of acetylated and tyrosinated microtubules, which represent stable and dynamic microtubules, respectively, in axons. Micro- tubules are densely packed in axon shafts, ...
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... us from resolving individual microtubules; therefore, we purposely ob- served specific portions of axons, where microtubules happened to be untied. Our observations showed a tendency for CAMSAP3 puncta to localize along a subpopulation of microtubules that are less acetylated, although this protein well coincided with tyrosinated microtubules (Fig. 3F, Left). In contrast, the distribu- tion of CAMSAP2 in relation to microtubule posttranscriptional modification was not so clear-cut as that of CAMSAP3. Unlike CAMSAP3, CAMSAP2 did not avoid acetylated microtubules (Fig. 3F, Right). Given that CAMSAP3 is localized to restricted zones in neurons (Fig. 1C), these observations suggest that, in ...
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... a subpopulation of microtubules that are less acetylated, although this protein well coincided with tyrosinated microtubules (Fig. 3F, Left). In contrast, the distribu- tion of CAMSAP2 in relation to microtubule posttranscriptional modification was not so clear-cut as that of CAMSAP3. Unlike CAMSAP3, CAMSAP2 did not avoid acetylated microtubules (Fig. 3F, Right). Given that CAMSAP3 is localized to restricted zones in neurons (Fig. 1C), these observations suggest that, in neurons, CAMSAP3 may affect tubulin acetylation only for a small fraction of microtubules and at levels not detectable using whole-cell ...
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Neurons are exquisitely polarized cells whose structure and function relies on microtubules. Microtubules in signal-receiving dendrites and signal-sending axons differ in their organization and microtubule-associated proteins. These differences, coupled with microtubule post-translational modifications, combine to locally regulate intracellular tra...
Citations
... 26 Subsequently, associated proteins, CAMSAP1, CAMSAP2, and CAMSAP3 have been identified, of which CAMSAP3 is identical to Nezha. [27][28][29][30] CAMSAP3 distributes in the apical region of epidermal cell, where it is responsible for establishing the apical-basal array of MTs. 27,31 Moreover, a pioneering study in Drosophila has demonstrated that patronin, also known as ssp4, is involved in the regulation of mitotic spindle assembly in S2 cells. ...
Synapses are fundamental components of the animal nervous system. Synaptic cytoskeleton is essential for maintaining proper neuronal development and wiring. Perturbations in neuronal microtubules (MTs) are correlated with numerous neuropsychiatric disorders. Despite discovering multiple synaptic MT regulators, the importance of MT stability, and particularly the polarity of MT in synaptic function, is still under investigation. Here, we identify Patronin, an MT minus-end-binding protein, for its essential role in presynaptic regulation of MT organization and neuromuscular junction (NMJ) development. Analyses indicate that Patronin regulates synaptic development independent of Klp10A. Subsequent research elucidates that it is short stop (Shot), a member of the Spectraplakin family of large cytoskeletal linker molecules, works synergistically with Patronin to govern NMJ development. We further raise the possibility that normal synaptic MT polarity contributes to proper NMJ morphology. Overall, this study demonstrates an unprecedented role of Patronin, and a potential involvement of MT polarity in synaptic development.
... How Ulk4 participates in these diverse processes has remained unclear. A recent study revealed that Ulk4 binds several other kinases and phosphatases such as DAPK3, LMTK2, PTPN14, as well as proteins implicated in the regulation of neuronal differentiation and axonal regeneration, including CAMSAP family proteins CAMSAP1/3 (Pongrakhananon et al., 2018;Preuss et al., 2020). Given our findings regarding how Ulk4 regulates Hh signaling, it is tempting to speculate that Ulk4 may also function as a scaffold to bridge kinases or other enzymes to their substrates and/or regulate the subcellular localization of the interacting proteins in other biological processes. ...
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that U nc-51- l ike k inase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.
... Loss of α-tubulin acetylation predisposes neurons to axon over-branching and excessive growth (Jenkins et al., 2017;Dan et al., 2018). The contribution of acetylated tubulin to neuronal morphogenesis was related to molecular effectors such as CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) which preferentially associates with non-acetylated MTs to regulate MT minus-end dynamics (Pongrakhananon et al., 2018) or to MT severing by katanin that preferentially occurs at acetylated MT sites in dendrites (Sudo and Baas, 2010). ...
... How Ulk4 participates in these diverse processes has remained unclear. A recent study revealed that Ulk4 binds several other kinases and phosphatases such as DAPK3, LMTK2, PTPN14, as well as proteins implicated in the regulation of neuronal differentiation and axonal regeneration, including CAMSAP family proteins CAMSAP1/3 (Pongrakhananon et al., 2018;Preuss et al., 2020). Given our findings regarding how Ulk4 regulates Hh signaling, it is tempting to speculate that Ulk4 may also function as a scaffold to bridge kinases or other enzymes to their substrates and/or regulate the subcellular localization of the interacting proteins in other biological processes. ...
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism remains poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.
... How Ulk4 participates in these diverse processes has remained unclear. A recent study revealed that Ulk4 binds several other kinases and phosphatases such as DAPK3, LMTK2, PTPN14 as well as proteins implicated in the regulation of neuronal differentiation and axonal regeneration including CAMSAP family proteins CAMSAP1/3 (Pongrakhananon et al., 2018 ;Preuss et al., 2020 ). Given our findings regarding how Ulk4 regulates Hh signaling, it is tempting to speculate that Ulk4 may also function as a scaffold to bridge kinases or other enzymes to their substrates, or regulate the subcellular localization of the interacting proteins in other biological processes. ...
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism remains poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.
... For instance, a protein, CAMSAP3 (in vertebrates), functions in concert with p60-katanin, and binds the minus-ends of microtubules, stabilizing them, and allowing tubulin polymerization at non-centrosomal sites to proceed (Figure 1; Pongrakhananon et al., 2018). ...
Katanin is a microtubule severing protein belonging to the ATPase family and consists of two subunits; p60-katanin synthesized by the KATNA1 gene and p80-katanin synthesized by the KATNB1 gene. Microtubule severing is one of the mechanisms that allow the reorganization of microtubules depending on cellular needs. While this reorganization of microtubules is associated with mitosis in dividing cells, it primarily takes part in the formation of structures such as axons and dendrites in nondividing mature neurons. Therefore, it is extremely important in neuronal branching. p60 and p80 katanin subunits coexist in the cell. While p60-katanin is responsible for cutting microtubules with its ATPase function, p80-katanin is responsible for the regulation of p60-katanin and its localization in the centrosome. Although katanin has vital functions in the cell, there are no known posttranscriptional regulators of it. MicroRNAs (miRNAs) are a group of small noncoding ribonucleotides that have been found to have important roles in regulating gene expression posttranscriptionally. Despite being important in gene regulation, so far no microRNA has been experimentally associated with katanin regulation. In this study, the effects of miR-124-3p, which we detected as a result of bioinformatics analysis to have the potential to bind to the p60 katanin mRNA, were investigated. For this aim, in this study, SH-SY5Y neuro-blastoma cells were transfected with pre-miR-124-3p mimics and pre-mir miRNA precursor as a negative control, and the effect of this transfection on p60-katanin expression was measured at both RNA and protein levels by quantitative real-time PCR (qRT-PCR) and western blotting, respectively. The results of this study showed for the first time that miR-124-3p, which was predicted to bind p60-katanin mRNA by bioinformatic analysis, may regulate the expression of the KATNA1 gene. The data obtained within the scope of this study will make important contributions in order to better understand the regulation of the expression of p60-katanin which as well will have an incontrovertible impact on the understanding of the importance of cytoskel-etal reorganization in both mitotic and postmitotic cells. K E Y W O R D S gene regulation, microtubule severing, miR-124-3p, p60-katanin
... These domains are thought to provide spectraplakins with elastic properties to respond to mechanical forces [34,48]. This domain also includes an Src-homology-3 (SH3) protein interaction domain, which is a structure that can bind several proteins such as the tumor suppressor p53, dynamin, a protein required for synaptic vesicle endocytosis, and CAMSAP3 (Calmodulin-regulated spectrin-associated protein family member 3), an MT-binding protein that is vital for proper neuronal growth [49][50][51]. The SH3 domain enables spectraplakins to interact with proteins (similar to p53, dynamin, and CAMSAP3) that are involved in cell migration, survival, scaffolding, vesicular transport, and cytoskeleton coordination [48,49,51]. ...
... This domain also includes an Src-homology-3 (SH3) protein interaction domain, which is a structure that can bind several proteins such as the tumor suppressor p53, dynamin, a protein required for synaptic vesicle endocytosis, and CAMSAP3 (Calmodulin-regulated spectrin-associated protein family member 3), an MT-binding protein that is vital for proper neuronal growth [49][50][51]. The SH3 domain enables spectraplakins to interact with proteins (similar to p53, dynamin, and CAMSAP3) that are involved in cell migration, survival, scaffolding, vesicular transport, and cytoskeleton coordination [48,49,51]. ...
... MACF1, through its SRD, can bind CAMSAP3 (calmodulinregulated spectrin-associated protein family member 3), a MT minus end protein important in MT stability [50]. CAMSAP3 is an essential protein in neuronal extension and dendrite growth, providing yet another mechanism for MACF1 to regulate transport [51]. Additionally, MACF1 plays a role in protein transport directly via interactions with the Golgi complex directly (via the PRD domain) and indirectly through its interaction with p230/GolginA4 [53,96]. ...
Bipolar affective disorder (BPAD) are life-long disorders that account for significant morbidity in afflicted patients. The etiology of BPAD is complex, combining genetic and environmental factors to increase the risk of disease. Genetic studies have pointed toward cytoskeletal dysfunction as a potential molecular mechanism through which BPAD may arise and have implicated proteins that regulate the cytoskeleton as risk factors. Microtubule actin crosslinking factor 1 (MACF1) is a giant cytoskeletal crosslinking protein that can coordinate the different aspects of the mammalian cytoskeleton with a wide variety of actions. In this review, we seek to highlight the functions of MACF1 in the nervous system and the molecular mechanisms leading to BPAD pathogenesis. We also offer a brief perspective on MACF1 and the role it may be playing in lithium's mechanism of action in treating BPAD.
... Patronin/CAMSAPs are also hypothesized to be non-centrosomal microtubule nucleators, thus providing an indispensable role in cells that need microtubules to be nucleated far from the cell centrosome (Meng et al, 2008;Goodwin & Vale, 2010;Tanaka et al, 2012;Feng et al, 2019;Imasaki et al, 2022). In cells, mutations or knockdown of CAMSAPs disturb microtubule growth or patterning, reaffirming the importance of these proteins in microtubule organization (Tanaka et al, 2012;Toya et al, 2016;Pongrakhananon et al, 2018;Coquand et al, 2021). ...
CAMSAPs are proteins that show microtubule minus-end–specific localization, decoration, and stabilization. Although the mechanism for minus-end recognition via their C-terminal CKK domain has been well described in recent studies, it is unclear how CAMSAPs stabilize microtubules. Our several binding assays revealed that the D2 region of CAMSAP3 specifically binds to microtubules with the expanded lattice. To investigate the relationship between this preference and the stabilization effect of CAMSAP3, we precisely measured individual microtubule lengths and found that D2 binding expanded the microtubule lattice by ∼3%. Consistent with the notion that the expanded lattice is a common feature of stable microtubules, the presence of D2 slowed the microtubule depolymerization rate to ∼1/20, suggesting that the D2-triggered lattice expansion stabilizes microtubules. Combining these results, we propose that CAMSAP3 stabilizes microtubules by lattice expansion upon D2 binding, which further accelerates the recruitment of other CAMSAP3 molecules. Because only CAMSAP3 has D2 and the highest microtubule-stabilizing effect among mammalian CAMSAPs, our model also explains the molecular basis for the functional diversity of CAMSAP family members.
... WDR47 has been reported to bind to the CAMSAP2-decorated microtubule, which is critical for the maintenance of CAMSAP2-positive microtubule stretch and prevents katanin-mediated severing of microtubule minus ends, thus ensuring proper stabilization of the microtubule network during neuronal development [111]. Neurons with CAM-SAP3 mutations have been found to generate multiple axons [112]. Mechanistically, CAMSAP3-bound microtubules cannot be acetylated, a process mediated by α-tubulin acetyltransferase-1 (ATAT1). ...
The establishment and maintenance of neuronal polarity are important for neural development and function. Abnormal neuronal polarity establishment commonly leads to a variety of neurodevelopmental disorders. Over the past three decades, with the continuous development and improvement of biological research methods and techniques, we have made tremendous progress in the understanding of the molecular mechanisms of neuronal polarity establishment. The activity of positive and negative feedback signals and actin waves are both essential in this process. They drive the directional transport and aggregation of key molecules of neuronal polarity, promote the spatiotemporal regulation of ordered and coordinated interactions of actin filaments and microtubules, stimulate the specialization and growth of axons, and inhibit the formation of multiple axons. In this review, we focus on recent advances in these areas, in particular the important findings about neuronal polarity in two classical models, in vitro primary hippocampal/cortical neurons and in vivo cortical pyramidal neurons, and discuss our current understanding of neuronal polarity.
... Our nocodazole washout experiments, in accordance with previously published results [28,29], showed that non-radial growth of microtubules became dominant in transfected cells. Interestingly, according to another study, the moderate overexpression of CAMSAP3 actively reduced tubulin acetylation in both epithelial and neuronal cells [35]. CAMSAP3 thus seems to serve the purpose of retaining a pool of dynamic microtubules in axons, although their microtubules are generally acetylated. ...
Membrane trafficking in interphase animal cells is accomplished mostly along the microtubules. Microtubules are often organized radially by the microtubule-organizing center to coordinate intracellular transport. Along with the centrosome, the Golgi often serves as a microtubule-organizing center, capable of nucleating and retaining microtubules. Recent studies revealed the role of a special subset of Golgi-derived microtubules, which facilitates vesicular traffic from this central transport hub of the cell. However, proteins essential for microtubule organization onto the Golgi might be differentially expressed in different cell lines, while many potential participants remain undiscovered. In the current work, we analyzed the involvement of the Golgi complex in microtubule organization in related cell lines. We studied two cell lines, both originating from green monkey renal epithelium, and found that they relied either on the centrosome or on the Golgi as a main microtubule-organizing center. We demonstrated that the difference in their Golgi microtubule-organizing activity was not associated with the well-studied proteins, such as CAMSAP3, CLASP2, GCC185, and GMAP210, but revealed several potential candidates involved in this process.
