Cytoplasmic Linker Protein Research Articles

CLASP-mediated competitive binding in protein condensates directs microtubule growth

Microtubule organization in cells relies on targeting mechanisms. Cytoplasmic linker proteins (CLIPs) and CLIP-associated proteins (CLASPs) are key regulators of microtubule organization, yet the underlying mechanisms remain elusive. Here, we reveal that the C-terminal domain of CLASP2 interacts with a common motif found in several CLASP-binding proteins. This interaction drives the dynamic localization of CLASP2 to distinct cellular compartments, where CLASP2 accumulates in protein condensates at the cell cortex or the microtubule plus end. These condensates physically contact each other via CLASP2-mediated competitive binding, determining cortical microtubule targeting. The phosphorylation of CLASP2 modulates the dynamics of the condensate-condensate interaction and spatiotemporally navigates microtubule growth. Moreover, we identify additional CLASP-interacting proteins that are involved in condensate contacts in a CLASP2-dependent manner, uncovering a general mechanism governing microtubule targeting. Our findings not only unveil a tunable multiphase system regulating microtubule organization, but also offer general mechanistic insights into intricate protein-protein interactions at the mesoscale level.

Autophagy initiation triggers p150Glued–AP-2β interaction on the lysosomes and facilitates their transport

The endocytic adaptor protein 2 (AP-2) complex binds dynactin as part of its noncanonical function, which is necessary for dynein-driven autophagosome transport along microtubules in neuronal axons. The absence of this AP-2-dependent transport causes neuronal morphology simplification and neurodegeneration. The mechanisms that lead to formation of the AP-2-dynactin complex have not been studied to date. However, the inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) enhances the transport of newly formed autophagosomes by influencing the biogenesis and protein interactions of Rab-interacting lysosomal protein (RILP), another dynein cargo adaptor. We tested effects of mTORC1 inhibition on interactions between the AP-2 and dynactin complexes, with a focus on their two essential subunits, AP-2β and p150Glued. We found that the mTORC1 inhibitor rapamycin enhanced p150Glued–AP-2β complex formation in both neurons and non-neuronal cells. Additional analysis revealed that the p150Glued–AP-2β interaction was indirect and required integrity of the dynactin complex. In non-neuronal cells rapamycin-driven enhancement of the p150Glued–AP-2β interaction also required the presence of cytoplasmic linker protein 170 (CLIP-170), the activation of autophagy, and an undisturbed endolysosomal system. The rapamycin-dependent p150Glued–AP-2β interaction occurred on lysosomal-associated membrane protein 1 (Lamp-1)-positive organelles but without the need for autolysosome formation. Rapamycin treatment also increased the acidification and number of acidic organelles and increased speed of the long-distance retrograde movement of Lamp-1-positive organelles. Altogether, our results indicate that autophagy regulates the p150Glued–AP-2β interaction, possibly to coordinate sufficient motor-adaptor complex availability for effective lysosome transport.

CLIP170 inhibits the metastasis and EMT of papillary thyroid cancer through the TGF-β pathway.

Metastasis poses a significant challenge in combating tumors. Even in papillary thyroid cancer (PTC), which typically exhibits a favorable prognosis, high recurrence rates are attributed to metastasis. Cytoplasmic linker protein 170 (CLIP170) functions as a classical microtubule plus-end tracking protein (+TIP) and has shown close association with cell migration. Nevertheless, the specific impact of CLIP170 on PTC cells remains to be elucidated. Our analysis of the GEO and TCGA databases unveiled an association between CLIP170 and the progression of PTC. To explore the impact of CLIP170 on PTC cells, we conducted various assays. We evaluated its effects through CCK-8, wound healing assay, and transwell assay after knocking down CLIP170. Additionally, the influence of CLIP170 on the cellular actin structure was examined via immunofluorescence; we further investigated the molecular expressions of epithelial-mesenchymal transition (EMT) and the transforming growth factor-β (TGF-β) signaling pathways through Western blotting and RT-qPCR. These findings were substantiated through an in vivo nude mouse model of lung metastasis. We observed a decreased expression of CLIP170 in PTC in contrast to normal thyroid tissue. Functionally, the knockdown of CLIP170 (CLIP170KD) notably enhanced the metastatic potential and EMT of PTC cells, both in vitro and in vivo. Mechanistically, CLIP170KD triggered the activation of the TGF-β pathway, subsequently promoting tumor cell migration, invasion, and EMT. Remarkably, the TGF-β inhibitor LY2157299 effectively countered TGF-β activity and significantly reversed tumor metastasis and EMT induced by CLIP170 knockdown. In summary, these findings collectively propose CLIP170 as a promising therapeutic target to mitigate metastatic tendencies in PTC.

LIS1 interacts with CLIP170 to promote tumor growth and metastasis via the Cdc42 signaling pathway in salivary gland adenoid cystic carcinoma.

Salivary gland adenoid cystic carcinoma (SACC) is one of the most common malignant tumors, with high aggressive potential in the oral and maxillofacial regions. Lissencephaly 1 (LIS1) is a microtubule-organizing center-associated protein that regulates the polymerization and stability of microtubules by mediating the motor function of dynein. Recent studies have suggested that LIS1 plays a potential role in the malignant development of tumors, such as in mitosis and migration. However, the role of LIS1 in SACC development and its related molecular mechanisms remain unclear. Thus, the effects of LIS1 on the proliferation, apoptosis, invasion and metastasis of SACC were studied, in vivo and in vitro. The results of immunohistochemical staining showed that LIS1 was highly expressed in SACC tissues, and its expression level was associated with malignant progression. In vitro, the results of CCK-8, TUNEL, wound healing and Transwell assays demonstrated that LIS1 promotes proliferation, inhibits apoptosis, and enhances the migration and invasion of SACC-LM cells. In vivo, knockdown of LIS1 effectively suppressed the growth of subcutaneous tumors in a mouse xenograft and distant metastasis of tumor cells in the metastasis model. The co-immunoprecipitation, immunofluorescence and western blot results also revealed that LIS1 binds to cytoplasmic linker protein 170 (CLIP170) to form a protein complex (LIS1/CLIP170), which activates the cell division control protein 42 homolog (Cdc42) signaling pathway to modulate the proliferation and anti-apoptosis of tumor cells, and enhanced invasion and metastasis by regulating the formation of invadopodia and the expression of MMPs in SACC-LM cells. Therefore, the present study demonstrated that LIS1 is a cancer promoter in SACC, and the molecular mechanism of the LIS1/CLIP170/Cdc42 signaling pathway is involved in the malignant progression, which offers a promising strategy for targeted therapy of SACC.

Pregnenolone-methyl-ether enhances CLIP170 and microtubule functions improving spine maturation and hippocampal deficits related to CDKL5 deficiency.

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170 (cytoplasmic linker protein 170). Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here, we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for 1 week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease-modifying therapies.

Dynactin 1 negatively regulates HIV-1 infection by sequestering the host cofactor CLIP170

Many viruses directly engage and require the dynein-dynactin motor-adaptor complex in order to transport along microtubules (MTs) to the nucleus and initiate infection. HIV type 1 (HIV-1) exploits dynein, the dynein adaptor BICD2, and core dynactin subunits but unlike several other viruses, does not require dynactin-1 (DCTN1). The underlying reason for HIV-1's variant dynein engagement strategy and independence from DCTN1 remains unknown. Here, we reveal that DCTN1 actually inhibits early HIV-1 infection by interfering with the ability of viral cores to interact with critical host cofactors. Specifically, DCTN1 competes for binding to HIV-1 particles with cytoplasmic linker protein 170 (CLIP170), one of several MT plus-end tracking proteins (+TIPs) that regulate the stability of viral cores after entry into the cell. Outside of its function as a dynactin subunit, DCTN1 also functions as a +TIP that we find sequesters CLIP170 from incoming particles. Deletion of the Zinc knuckle (Zn) domain in CLIP170 that mediates its interactions with several proteins, including DCTN1, increased CLIP170 binding to virus particles but failed to promote infection, further suggesting that DCTN1 blocks a critical proviral function of CLIP170 mediated by its Zn domain. Our findings suggest that the unique manner in which HIV-1 binds and exploits +TIPs to regulate particle stability leaves them vulnerable to the negative effects of DCTN1 on +TIP availability and function, which may in turn have driven HIV-1 to evolve away from DCTN1 in favor of BICD2-based engagement of dynein during early infection.

HIV-1 capsids mimic a microtubule regulator to coordinate early stages of infection.

While the microtubule end-binding protein, EB1 facilitates early stages of HIV-1 infection, how it does so remains unclear. Here, we show that beyond its effects on microtubule acetylation, EB1 also indirectly contributes to infection by delivering the plus-end tracking protein (+TIP), cytoplasmic linker protein 170 (CLIP170) to the cell periphery. CLIP170 bound to intact HIV-1 cores or invitro assembled capsid-nucleocapsid complexes, while EB1 did not. Moreover, unlike EB1 and several other +TIPs, CLIP170 enhanced infection independently of effects on microtubule acetylation. Capsid mutants and imaging revealed that CLIP170 bound HIV-1 cores in a manner distinct from currently known capsid cofactors, influenced by pentamer composition or curvature. Structural analyses revealed an EB-like +TIP-binding motif within the capsid major homology region (MHR) that binds SxIP motifs found in several +TIPs, and variability across this MHR sequence correlated with the extent to which different retroviruses engage CLIP170 to facilitate infection. Our findings provide mechanistic insights into the complex roles of +TIPs in mediating early stages of retroviral infection, and reveal divergent capsid-based EB1 mimicry across retroviral species.

Arsenic trioxide disturbs the LIS1/NDEL1/dynein microtubule dynamic complex by disrupting the CLIP170 zinc finger in head and neck cancer

Cancer mortality is mainly caused by metastasis, which requires dynamic remodeling of cytoskeletal components such as microtubules. Targeting microtubules presents a promising antimetastatic strategy that could prevent cancer spreading and recurrence. It is known that arsenic trioxide (ATO) is able to inhibit the migration and invasion of solid malignant tumors, but its exact molecular mechanism remains unclear. Here, we report a novel molecular target and antimetastatic mechanism of ATO in head and neck squamous cell carcinoma (HNSCC). We found that cytoplasmic linker protein 170 (CLIP170) was overexpressed in HNSCC tissues and cells compared to normal controls. ATO at non-cytotoxic level (1 μM) inhibited the migration and invasion of HNSCC cells by displacing zinc in the zinc finger motif of CLIP170, which is a key protein that controls microtubule dynamics. The antimetastatic effects of ATO were equivalent to those of siRNA-mediated CLIP170 knockdown. Furthermore, ATO dysregulated microtubule polymerization via the CLIP170/LIS1/NDEL1/dynein signaling pathway in Cal27 cells as a functional consequence of CLIP170 zinc finger disruption. The effect was partially reversed by zinc supplementation. Taken together, these findings reveal that CLIP170 is a novel molecular target of ATO and demonstrate the capability and underlying mechanisms of ATO as a potential antimetastatic agent for HNSCC treatment.

Exogenous CLASP2 protein treatment enhances wound healing in vitro and in vivo.

Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3β, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.

The neurosteroid pregnenolone promotes degradation of key proteins in the innate immune signaling to suppress inflammation

Pregnenolone is a steroid hormone precursor that is synthesized in various steroidogenic tissues, in the brain, and in lymphocytes. In addition to serving as the precursor for other steroid hormones, pregnenolone exerts its own effect as an anti-inflammatory molecule to maintain immune homeostasis in various inflammatory conditions. Pregnenolone and its metabolic derivatives have been shown to have beneficial effects in the brain, including enhancing memory and learning, reversing depressive disorders, and modulating cognitive functions. A decreased level of pregnenolone has been observed in neuroinflammatory diseases, which emphasizes its role in neuroprotection and neuroregeneration. Although the anti-inflammatory property of pregnenolone was recognized several decades ago, its mechanism of action remains unknown. Here we report that pregnenolone promotes ubiquitination and degradation of the TLR2/4 adaptor protein TIRAP and TLR2 in macrophages and microglial cells. Pregnenolone and its metabolites suppressed the secretion of tumor necrosis factor α and interleukin-6 mediated through TLR2 and TLR4 signaling. Pregnenolone has been reported to induce activation of cytoplasmic linker protein 170, and this protein has recently been shown to promote targeted degradation of TIRAP. We observed enhanced degradation of TIRAP and TLR4 suppression by cytoplasmic linker protein 170 in the presence of pregnenolone. Our experimental data reveal novel nongenomic targets of pregnenolone and provide important leads to understand its role in restoring immune homeostasis in various inflammatory conditions.

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

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

CLIP-170 is essential for MTOC repositioning during T cell activation by regulating dynein localisation on the cell surface

The microtubule-organizing centre (MTOC) is repositioned to the centre of the contacted cell surface, the immunological synapse, during T cell activation. However, our understanding of its molecular mechanism remains limited. Here, we found that the microtubule plus-end tracking cytoplasmic linker protein 170 (CLIP-170) plays a novel role in MTOC repositioning using fluorescence imaging. Inhibition of CLIP-170 phosphorylation impaired both MTOC repositioning and interleukin-2 (IL-2) expression. T cell stimulation induced some fraction of dynein to colocalise with CLIP-170 and undergo plus-end tracking. Concurrently, it increased dynein in minus-end-directed movement. It also increased dynein relocation to the centre of the contact surface. Dynein not colocalised with CLIP-170 showed both an immobile state and minus-end-directed movement at a velocity in good agreement with the velocity of MTOC repositioning, which suggests that dynein at the immunological synapse may pull the microtubules and the MTOC. Although CLIP-170 is phosphorylated by AMP-activated protein kinase (AMPK) irrespective of stimulation, phosphorylated CLIP-170 is essential for dynein recruitment to plus-end tracking and for dynein relocation. This indicates that dynein relocation results from coexistence of plus-end- and minus-end-directed translocation. In conclusion, CLIP-170 plays an indispensable role in MTOC repositioning and full activation of T cells by regulating dynein localisation.

The Globodera pallida SPRYSEC Effector GpSPRY-414-2 That Suppresses Plant Defenses Targets a Regulatory Component of the Dynamic Microtubule Network.

The white potato cyst nematode, Globodera pallida, is an obligate biotrophic pathogen of a limited number of Solanaceous plants. Like other plant pathogens, G. pallida deploys effectors into its host that manipulate the plant to the benefit of the nematode. Genome analysis has led to the identification of large numbers of candidate effectors from this nematode, including the cyst nematode-specific SPRYSEC proteins. These are a secreted subset of a hugely expanded gene family encoding SPRY domain-containing proteins, many of which remain to be characterized. We investigated the function of one of these SPRYSEC effector candidates, GpSPRY-414-2. Expression of the gene encoding GpSPRY-414-2 is restricted to the dorsal pharyngeal gland cell and reducing its expression in G. pallida infective second stage juveniles using RNA interference causes a reduction in parasitic success on potato. Transient expression assays in Nicotiana benthamiana indicated that GpSPRY-414-2 disrupts plant defenses. It specifically suppresses effector-triggered immunity (ETI) induced by co-expression of the Gpa2 resistance gene and its cognate avirulence factor RBP-1. It also causes a reduction in the production of reactive oxygen species triggered by exposure of plants to the bacterial flagellin epitope flg22. Yeast two-hybrid screening identified a potato cytoplasmic linker protein (CLIP)-associated protein (StCLASP) as a host target of GpSPRY-414-2. The two proteins co-localize in planta at the microtubules. CLASPs are members of a conserved class of microtubule-associated proteins that contribute to microtubule stability and growth. However, disruption of the microtubule network does not prevent suppression of ETI by GpSPRY-414-2 nor the interaction of the effector with its host target. Besides, GpSPRY-414-2 stabilizes its target while effector dimerization and the formation of high molecular weight protein complexes including GpSPRY-414-2 are prompted in the presence of the StCLASP. These data indicate that the nematode effector GpSPRY-414-2 targets the microtubules to facilitate infection.

Cytoplasmic Linker Protein CLIP170 Negatively Regulates TLR4 Signaling by Targeting the TLR Adaptor Protein TIRAP.

Cytoplasmic linker protein 170 (CLIP170) is a CAP-Gly domain-containing protein that is associated with the plus end of growing microtubules and implicated in various cellular processes, including the regulation of microtubule dynamics, cell migration, and intracellular transport. Our studies revealed a previously unrecognized property and role of CLIP170. We identified CLIP170 as one of the interacting partners of Brucella effector protein TcpB that negatively regulates TLR2 and TLR4 signaling. In this study, we demonstrate that CLIP170 interacts with the TLR2 and TLR4 adaptor protein TIRAP. Furthermore, our studies revealed that CLIP170 induces ubiquitination and subsequent degradation of TIRAP to negatively regulate TLR4-mediated proinflammatory responses. Overexpression of CLIP170 in mouse macrophages suppressed the LPS-induced expression of IL-6 and TNF-α whereas silencing of endogenous CLIP170 potentiated the levels of proinflammatory cytokines. In vivo silencing of CLIP170 in C57BL/6 mice by CLIP170-specific small interfering RNA enhanced LPS-induced IL-6 and TNF-α expression. Furthermore, we found that LPS modulates the expression of CLIP170 in mouse macrophages. Overall, our experimental data suggest that CLIP170 serves as an intrinsic negative regulator of TLR4 signaling that targets TIRAP.

Phosphorylation of EB1 regulates the recruitment of CLIP-170 and p150glued to the plus ends of astral microtubules.

Phosphorylation of end-binding protein 1 (EB1), a key member of microtubule plus end-tracking proteins (+TIPs), by apoptosis signal-regulating kinase 1 (ASK1) has been demonstrated to promote the stability of astral microtubules during mitosis by stimulating the binding of EB1 to microtubule plus ends. However, the roles of other members of the +TIPs family in ASK1/EB1-mediated regulation of astral microtubules are unknown. Herein, we show that ASK1-mediated phosphorylation of EB1 enhances the localization of cytoplasmic linker protein 170 (CLIP-170) and p150glued to the plus ends of astral microtubules. Depletion of ASK1 or expression of phospho-deficient or phospho-mimetic EB1 mutants results in changes in the levels of plus-end localized CLIP-170 or p150glued. Mechanistic studies reveal that EB1 phosphorylation promotes its interactions with CLIP-170 and p150glued, thereby recruiting these +TIPs to microtubules. Structural analysis suggests that serine-40 is the primary phosphorylation site on EB1 that exerts these effects. Together, these findings provide novel insight into the molecular mechanisms that regulate the interactions of EB1 with other +TIPs.

Microtubule plus end-associated CLIP-170 initiates HSV-1 retrograde transport in primary human cells.

Dynamic microtubules (MTs) continuously explore the intracellular environment and, through specialized plus end-tracking proteins (+TIPs), engage a variety of targets. However, the nature of cargoes that require +TIP-mediated capture for their movement on MTs remains poorly understood. Using RNA interference and dominant-negative approaches, combined with live cell imaging, we show that herpes simplex virus particles that have entered primary human cells exploit a +TIP complex comprising end-binding protein 1 (EB1), cytoplasmic linker protein 170 (CLIP-170), and dynactin-1 (DCTN1) to initiate retrograde transport. Depletion of these +TIPs completely blocked post-entry long-range transport of virus particles and suppressed infection ∼5,000-fold, whereas transferrin uptake, early endosome organization, and dynein-dependent movement of lysosomes and mitochondria remained unaffected. These findings provide the first insights into the earliest stages of viral engagement of MTs through specific +TIPs, akin to receptors, with therapeutic implications, and identify herpesvirus particles as one of a very limited number of cargoes absolutely dependent on CLIP-170-mediated capture to initiate transport in primary human cells.

Identification of a Novel Sequence Motif Recognized by the Ankyrin Repeat Domain of zDHHC17/13 S-Acyltransferases

S-Acylation is a major post-translational modification affecting several cellular processes. It is particularly important for neuronal functions. This modification is catalyzed by a family of transmembrane S-acyltransferases that contain a conserved zinc finger DHHC (zDHHC) domain. Typically, eukaryote genomes encode for 7-24 distinct zDHHC enzymes, with two members also harboring an ankyrin repeat (AR) domain at their cytosolic N termini. The AR domain of zDHHC enzymes is predicted to engage in numerous interactions and facilitates both substrate recruitment and S-acylation-independent functions; however, the sequence/structural features recognized by this module remain unknown. The two mammalian AR-containing S-acyltransferases are the Golgi-localized zDHHC17 and zDHHC13, also known as Huntingtin-interacting proteins 14 and 14-like, respectively; they are highly expressed in brain, and their loss in mice leads to neuropathological deficits that are reminiscent of Huntington's disease. Here, we report that zDHHC17 and zDHHC13 recognize, via their AR domain, evolutionary conserved and closely related sequences of a [VIAP][VIT]XXQP consensus in SNAP25, SNAP23, cysteine string protein, Huntingtin, cytoplasmic linker protein 3, and microtubule-associated protein 6. This novel AR-binding sequence motif is found in regions predicted to be unstructured and is present in a number of zDHHC17 substrates and zDHHC17/13-interacting S-acylated proteins. This is the first study to identify a motif recognized by AR-containing zDHHCs.

CLIP-170 tethers kinetochores to microtubule plus ends against poleward force by dynein for stable kinetochore–microtubule attachment

The cytoplasmic linker protein (CLIP)-170 localizes to kinetochores and is suggested to function in stable attachment of kinetochores to microtubule ends. Here we show that defects in kinetochore–microtubule attachment and chromosome alignment in CLIP-170-depleted cells were rescued by co-depletion of p150glued, a dynactin subunit required for kinetochore localization of CLIP-170. CLIP-170 recruited p150glued to microtubule ends. Kinetochore localization at microtubule ends was perturbed by CLIP-170 depletion, which was rescued by co-depleting p150glued. Our results imply that CLIP-170 tethers kinetochores to microtubule ends against the dynein-mediated poleward force to slide kinetochores along microtubules, facilitating the stable kinetochore attachment to microtubules.

Hook proteins: association with Alzheimer pathology and regulatory role of hook3 in amyloid beta generation.

Defects in intracellular transport are implicated in the pathogenesis of Alzheimer’s disease (AD). Hook proteins are a family of cytoplasmic linker proteins that participate in endosomal transport. In this study we show that Hook1 and Hook3 are expressed in neurons while Hook2 is predominantly expressed in astrocytes. Furthermore, Hook proteins are associated with pathological hallmarks in AD; Hook1 and Hook3 are localized to tau aggregates and Hook2 to glial components within amyloid plaques. Additionally, the expression of Hook3 is reduced in AD. Modelling of Hook3 deficiency in cultured cells leads to slowing of endosomal transport and increases β-amyloid production. We propose that Hook3 plays a role in pathogenic events exacerbating AD.

HookA is a novel dynein–early endosome linker critical for cargo movement in vivo

Cytoplasmic dynein transports membranous cargoes along microtubules, but the mechanism of dynein-cargo interaction is unclear. From a genetic screen, we identified a homologue of human Hook proteins, HookA, as a factor required for dynein-mediated early endosome movement in the filamentous fungus Aspergillus nidulans. HookA contains a putative N-terminal microtubule-binding domain followed by coiled-coil domains and a C-terminal cargo-binding domain, an organization reminiscent of cytoplasmic linker proteins. HookA-early endosome interaction occurs independently of dynein-early endosome interaction and requires the C-terminal domain. Importantly, HookA interacts with dynein and dynactin independently of HookA-early endosome interaction but dependent on the N-terminal part of HookA. Both dynein and the p25 subunit of dynactin are required for the interaction between HookA and dynein-dynactin, and loss of HookA significantly weakens dynein-early endosome interaction, causing a virtually complete absence of early endosome movement. Thus, HookA is a novel linker important for dynein-early endosome interaction in vivo.