Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.

A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

Bluetongue virus infection induces aberrant mitosis in mammalian cells.

BACKGROUND: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division. METHODS: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2. RESULTS: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects. CONCLUSIONS: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.

Mutation of the variant alpha-tubulin TUBA8 results in polymicrogyria with optic nerve hypoplasia.

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

Cytoskeletal analysis of human blastocysts by confocal laser scanning microscopy following vitrification.

BACKGROUND: Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS: A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS: In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9%) were normal, 20/213 (9.4%) were abnormally shaped, 9/213 (4.2%) were multipolar and 1/213 (0.5%) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6%) were normal, 39/175 (22.3%) were abnormally shaped, 10/175 (5.7%) were multipolar and 6/175 (3.4%) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS: The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts.

A simple cell based assay to measure Parkin activity.

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin ß-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.

Evaluation of institutional readiness at sites within the UK NHS using a novel advanced therapy medicinal product assessment tool.

Aim: This paper reports on a study to assess institutional readiness (IR) of UK National Health Service sites that form part of the Northern Alliance Advanced Therapy Treatment Centre to deliver advanced therapy medicinal products (ATMPs). The paper discusses the development of an assessment tool to support self-assessment of IR in healthcare institutions. Methods: The tool utilized criteria developed by clinicians to self-assess IR to deliver four classes of ATMP over a series of time points. Each assessment was independently analyzed and validated by independent expert groups. Results & conclusion: The collated results indicated an overall trend toward IR for all classes of ATMP. The study highlighted areas where IR is evidenced, areas where work is ongoing and areas where further work is required to achieve IR. The study also facilitated validation of the IR assessment tool.

Tweetable abstract This paper describes the development of a novel tool for assessment of healthcare institutions' capacity to deliver advanced therapies and use of the tool to evaluate institutional readiness within selected UK National Health Service sites. #advancedtherapies @NAATTC @innovateuk.

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis.

BACKGROUND: Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). RESULTS: We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. CONCLUSIONS: These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.

Bladder tumour-derived somatic TSC1 missense mutations cause loss of function via distinct mechanisms.

More than 50% of transitional cell carcinomas of the bladder show loss of heterozygosity of a region spanning the TSC1 locus at 9q34 and mutations of TSC1 have been identified in 14.5% of tumours. These comprise nonsense mutations, splicing mutations, small deletions and missense mutations. Missense mutations are only rarely found in the germline in TSC disease. Therefore, we have examined six somatic missense mutations found in bladder cancer to determine whether these result in loss of function. We describe loss of function via distinct mechanisms. Five mutations caused mutually exclusive defects at mRNA and protein levels. Of these, two mutations caused pre-mRNA splicing errors that were predicted to result in premature protein truncation and three resulted in markedly reduced stability of exogenous TSC1 protein. Primary tumours with aberrant TSC1 pre-mRNA splicing were confirmed as negative for TSC1 expression by immunohistochemistry. Expression was also significantly reduced in a tumour with a TSC1 missense mutation resulting in diminished protein half-life. A single TSC1 missense mutation identified in a tumour with retained heterozygosity of the TSC1 region on chromosome 9 caused an apparently TSC2- and mTOR-independent localization defect of the mutant protein. We conclude that although TSC1 missense mutations do not play a major role in causation of TSC disease, they represent a significant proportion of somatic loss of function mutations in bladder cancer.

Tumor infection by oncolytic reovirus primes adaptive antitumor immunity.

PURPOSE: Early clinical trials are under way exploring the direct oncolytic potential of reovirus. This study addresses whether tumor infection by reovirus is also able to generate bystander, adaptive antitumor immunity. EXPERIMENTAL DESIGN: Reovirus was delivered intravenously to C57BL/6 mice bearing lymph node metastases from the murine melanoma, B16-tk, with assessment of nodal metastatic clearance, priming of antitumor immunity against the tumor-associated antigen tyrosinase-related protein-2, and cytokine responses. In an in vitro human system, the effect of reovirus infection on the ability of Mel888 melanoma cells to activate and load dendritic cells for cytotoxic lymphocyte (CTL) priming was investigated. RESULTS: In the murine model, a single intravenous dose of reovirus reduced metastatic lymph node burden and induced antitumor immunity (splenocyte response to tyrosinase-related protein-2 and interleukin-12 production in disaggregated lymph nodes). In vitro human assays revealed that uninfected Mel888 cells failed to induce dendritic cell maturation or support priming of an anti-Mel888 CTL response. In contrast, reovirus-infected Mel888 cells (reo-Mel) matured dendritic cells in a reovirus dose-dependent manner. When cultured with autologous peripheral blood lymphocytes, dendritic cells loaded with reo-Mel induced lymphocyte expansion, IFN-gamma production, specific anti-Mel888 cell cytotoxicity, and cross-primed CD8+ T cells specific against the human tumor-associated antigen MART-1. CONCLUSION: Reovirus infection of tumor cells reduces metastatic disease burden and primes antitumor immunity. Future clinical trials should be designed to explore both direct cytotoxic and immunotherapeutic effects of reovirus.

The APC-EB1 interaction.

Changes in cell proliferation seen in cancers initiated by adenomatous polyposis coli (APC) gene mutation are driven by the loss of an ability to negatively regulate the canonical WNT signalling pathway. However, mutant APC proteins also lack the ability to interact with a number of other ligands and it is possible that the loss of these interactions could contribute to the phenotype or to the development ofcolorectal tumours. One such association is with the microtubule plus-end binding protein EB1. Originally identified as an APC binding partner, EB1 is now known to be part of an evolutionarily conserved family of proteins involved in the regulation of microtubule dynamics and microtubule-dependent processes. Roles for the interaction between APC and EB1 have been identified in cellular functions as diverse as directed cell migration and mitosis, with potentially important implications for the behaviour of both normal epithelial cells and colorectal cancer cells. In this chapter our current understanding of the functional role of the APC-EB1 interaction will be reviewed.

A MAPK/c-Jun-mediated switch regulates the initial adaptive and cell death responses to mitochondrial damage in a neuronal cell model.

Parkinson's disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor c-Jun can act in either a pro- or anti-apoptotic manner, depending on the level of stress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategies.

Expression analysis of the MCPH1/BRIT1 and BRCA1 tumor suppressor genes and telomerase splice variants in epithelial ovarian cancer.

AIMS: The aim of this study was to explore the correlation of hTERT splice variant expression with MCPH1/BRIT1 and BRCA1 expression in epithelial ovarian cancer (EOC) samples. BACKGROUND: Telomerase activation can contribute to the progression of tumors and the development of cancer. However, the regulation of telomerase activity remains unclear. MCPH1 (also known as BRIT1, BRCT-repeat inhibitor of hTERT expression) and BRCA1 are tumor suppressor genes that have been linked to telomerase expression. METHODS: qPCR was used to investigate telomerase splice variants, MCPH1/BRIT1 and BRCA1 expression in EOC tissue and primary cultures. RESULTS: The wild type α+/ß+ hTERT variant was the most common splice variant in the EOC samples, followed by α+/ß- hTERT, a dominant negative regulator of telomerase activity. EOC samples expressing high total hTERT demonstrated significantly lower MCPH1/BRIT1 expression in both tissue (p = 0.05) and primary cultures (p = 0.03). We identified a negative correlation between MCPH1/BRIT1 and α+/ß+ hTERT (p = 0.04), and a strong positive association between MCPH1/BRIT1 and both α-/ß+ hTERT and α-/ß- hTERT (both p = 0.02). A positive association was observed between BRCA1 and α-/ß+ hTERT and α-/ß- hTERT expression (p = 0.003 and p = 0.04, respectively). CONCLUSIONS: These findings support a regulatory effect of MCPH1/BRIT1 and BRCA1 on telomerase activity, particularly the negative association between MCPH1/BRIT1 and the functional form of hTERT (α+/ß+).

Loss of CSMD1 expression disrupts mammary duct formation while enhancing proliferation, migration and invasion.

The CUB and sushi multiple domains 1 (CSMD1) gene maps to chromosome 8p23, a region deleted in many cancers. Loss of CSMD1 expression is associated with poor prognosis in breast cancer suggesting that it acts as a tumour suppressor in this cancer. However, the function of CSMD1 is largely unknown. Herein, we investigated CSMD1 functions in cell line models. CSMD1 expression was suppressed in MCF10A and LNCaP cells using short hairpin RNA. Functional assays were performed focusing on the 'normal' MCF10A cell line. Suppression of CSMD1 significantly increased the proliferation, cell migration and invasiveness of MCF10A cells compared to shcontrols. shCSMD1 cells also showed significantly reduced adhesion to Matrigel and fibronectin. In a three-dimensional Matrigel model of MCF10A cells, reduced CSMD1 expression resulted in the development of larger and more poorly differentiated breast acini-like structures that displayed impaired lumen formation. Loss of CSMD1 expression disrupts a model of mammary duct formation while enhancing proliferation, migration and invasion. Our data suggest that CSMD1 is involved in the suppression of a transformed phenotype.

Oncolytic Herpes Simplex Virus Inhibits Pediatric Brain Tumor Migration and Invasion.

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are invasive tumors with poor survival. Oncolytic virotherapy, initially devised as a direct cytotoxic treatment, is now also known to act via immune-mediated mechanisms. Here we investigate a previously unreported mechanism of action: the inhibition of migration and invasion in pediatric brain tumors. We evaluated the effect of oncolytic herpes simplex virus 1716 (HSV1716) on the migration and invasion of pHGG and DIPG both in vitro using 2D (scratch assay, live cell imaging) and 3D (spheroid invasion in collagen) assays and in vivo using an orthotopic xenograft model of DIPG invasion. HSV1716 inhibited migration and invasion in pHGG and DIPG cell lines. pHGG cells demonstrated reduced velocity and changed morphology in the presence of virus. HSV1716 altered pHGG cytoskeletal dynamics by stabilizing microtubules, inhibiting glycogen synthase kinase-3, and preventing localized clustering of adenomatous polyposis coli (APC) to the leading edge of cells. HSV1716 treatment also reduced tumor infiltration in a mouse orthotopic xenograft DIPG model. Our results demonstrate that HSV1716 targets the migration and invasion of pHGG and DIPG and indicates the potential of an oncolytic virus (OV) to be used as a novel anti-invasive treatment strategy for pediatric brain tumors.

A tubulin alpha 8 mouse knockout model indicates a likely role in spermatogenesis but not in brain development.

Tubulin alpha 8 (Tuba8) is the most divergent member of the highly conserved alpha tubulin family, and uniquely lacks two key post-translational modification sites. It is abundantly expressed in testis and muscle, with lower levels in the brain. We previously identified homozygous hypomorphic TUBA8 mutations in human subjects with a polymicrogyria (PMG) syndrome, suggesting its involvement in development of the cerebral cortex. We have now generated and characterized a Tuba8 knockout mouse model. Homozygous mice were confirmed to lack Tuba8 protein in the testis, but did not display PMG and appeared to be neurologically normal. In response to this finding, we re-analyzed the human PMG subjects using whole exome sequencing. This resulted in identification of an additional homozygous loss-of-function mutation in SNAP29, suggesting that SNAP29 deficiency, rather than TUBA8 deficiency, may underlie most or all of the neurodevelopmental anomalies in these subjects. Nonetheless, in the mouse brain, Tuba8 specifically localised to the cerebellar Purkinje cells, suggesting that the human mutations may affect or modify motor control. In the testis, Tuba8 localisation was cell-type specific. It was restricted to spermiogenesis with a strong acrosomal localization that was gradually replaced by cytoplasmic distribution and was absent from spermatozoa. Although the knockout mice were fertile, the localisation pattern indicated that Tuba8 may have a role in spermatid development during spermatogenesis, rather than as a component of the mature microtubule-rich flagellum itself.

Examination of actin and microtubule dependent APC localisations in living mammalian cells.

BACKGROUND: The trafficking of the adenomatous polyposis coli (APC) tumour suppressor protein in mammalian cells is a perennially controversial topic. Immunostaining evidence for an actin-associated APC localisation at intercellular junctions has been previously presented, though live imaging of mammalian junctional APC has not been documented. RESULTS: Using live imaging of transfected COS-7 cells we observed intercellular junction-associated pools of GFP-APC in addition to previously documented microtubule-associated GFP-APC and a variety of minor localisations. Although both microtubule and junction-associated populations could co-exist within individual cells, they differed in their subcellular location, dynamic behaviour and sensitivity to cytoskeletal poisons. GFP-APC deletion mutant analysis indicated that a protein truncated immediately after the APC armadillo repeat domain retained the ability to localise to adhesive membranes in transfected cells. Supporting this, we also observed junctional APC immunostaining in cultures of human colorectal cancer cell line that express truncated forms of APC. CONCLUSION: Our data indicate that APC can be found in two spatially separate populations at the cell periphery and these populations can co-exist in the same cell. The first localisation is highly dynamic and associated with microtubules near free edges and in cell vertices, while the second is comparatively static and is closely associated with actin at sites of cell-cell contact. Our imaging confirms that human GFP-APC possesses many of the localisations and behaviours previously seen by live imaging of Xenopus GFP-APC. However, we report the novel finding that GFP-APC puncta can remain associated with the ends of shrinking microtubules. Deletion analysis indicated that the N-terminal region of the APC protein mediated its junctional localisation, consistent with our observation that truncated APC proteins in colon cancer cell lines are still capable of localising to the cell cortex. This may have implications for the development of colorectal cancer.

Trapping of normal EB1 ligands in aggresomes formed by an EB1 deletion mutant.

BACKGROUND: EB1 is a microtubule tip-associated protein that interacts with the APC tumour suppressor protein and the p150glued subunit of dynactin. We previously reported that an EB1 deletion mutant that retains both of these interactions but does not directly associate with microtubules (EB1-DeltaN2-GFP) spontaneously formed perinuclear aggregates when expressed in COS-7 cells. RESULTS: In the present study live imaging indicated that EB1-DeltaN2-GFP aggregates underwent dynamic microtubule-dependent changes in morphology and appeared to be internally cohesive. EB1-DeltaN2-GFP aggregates were phase-dense structures that displayed microtubule-dependent accumulation around the centrosome, were immunoreactive for both the 20s subunit of the proteasome and ubiquitin, and induced the collapse of the vimentin cytoskeleton. Fractionation studies revealed that a proportion of EB1-DeltaN2-GFP was detergent-insoluble and ubiquitylated, indicating that EB1-DeltaN2-GFP aggregates are aggresomes. Immunostaining also revealed that APC and p150glued were present in EB1-DeltaN2-GFP aggregates, whereas EB3 was not. Furthermore, evidence for p150glued degradation was found in the insoluble fraction of EB1-DeltaN2-GFP transfected cultures. CONCLUSION: Our data indicate that aggresomes can be internally cohesive and may not represent a simple "aggregate of aggregates" assembled around the centrosome. Our observations also indicate that a partially misfolded protein may retain the ability to interact with its normal physiological ligands, leading to their co-assembly into aggresomes. This supports the idea that the trapping and degradation of co-aggregated proteins might contribute to human pathologies characterised by aggresome formation.

The Leber congenital amaurosis protein AIPL1 and EB proteins co-localize at the photoreceptor cilium.

PURPOSE: The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1). METHODS: The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy. RESULTS: Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous ß-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors. CONCLUSIONS: AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.