Tubulin Folding Research Articles

Visualizing nucleation, condensation and propagation of β-tubulin folding in chaperonin TRiC.

The folding nucleus (FN) initiates protein folding and enables an efficient folding pathway. Here we directly visualize the tubulin FN consisting of a nonnative, partially assembled Rossmann fold, in the closed chamber of human chaperonin TRiC. Chaperonin TRiC interacts with non-natively folded secondary structural elements, stabilizing the nucleus for transition into its first native domain. Through progressive folding, the unfolded sequence goes through drastic spatial arrangement in the TRiC chamber to sample the conformational space, mediated by the highly dynamic CCT tails. The observed presence of individual nonnative secondary structures first in the nonnative FN and then around the incrementally folded native domains supports the hypothesis that tubulin folding in TRiC is a hierarchical process of nucleation, condensation and propagation in cooperation with TRiC subunits.

Lassa virus Z protein hijacks the autophagy machinery for efficient transportation by interrupting CCT2-mediated cytoskeleton network formation

ABSTRACT The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation. Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4’,6-diamidino-2’-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole.

The CCTδ subunit of the molecular chaperone CCT is required for correct localisation of p150Glued to spindle poles during mitosis

Chaperonin Containing Tailless complex polypeptide 1 (CCT) is a molecular chaperone composed of eight distinct subunits that can exist as individual monomers or as components of a double oligomeric ring, which is essential for the folding of actin and tubulin and other substrates. Here we assess the role of CCT subunits in the context of cell cycle progression by individual subunit depletions upon siRNA treatment in mammalian cells. The depletion of individual CCT subunits leads to variation in the distribution of cell cycle phases and changes in mitotic index. Mitotic defects, such as unaligned chromosomes occur when CCTδ is depleted, concurrent with a reduction in spindle pole-localised p150Glued, a component of the dynactin complex and a binding partner of monomeric CCTδ. In CCTδ-depleted cells, changes in the elution profile of p150Glued are observed consistent with altered conformations and or assembly states with the dynactin complex. Addition of monomeric CCTδ, in the form of GFP-CCTδ, restores correct p150Glued localisation to the spindle poles and rescues the mitotic segregation defects that occur when CCTδ is depleted. This study demonstrates a requirement for CCTδ in its monomeric form for correct chromosome segregation via a mechanism that promotes the correct localisation of p150Glued, thus revealing further complexities to the interplay between CCT, tubulin folding and microtubule dynamics.

STYXL1 regulates CCT complex assembly and flagellar tubulin folding in sperm formation

Tubulin-based microtubule is a core component of flagella axoneme and essential for sperm motility and male fertility. Structural components of the axoneme have been well explored. However, how tubulin folding is regulated in sperm flagella formation is still largely unknown. Here, we report a germ cell-specific co-factor of CCT complex, STYXL1. Deletion of Styxl1 results in male infertility and microtubule defects of sperm flagella. Proteomic analysis of Styxl1-/- sperm reveals abnormal downregulation of flagella-related proteins including tubulins. The N-terminal rhodanese-like domain of STYXL1 is important for its interactions with CCT complex subunits, CCT1, CCT6 and CCT7. Styxl1 deletion leads to defects in CCT complex assembly and tubulin polymerization. Collectively, our findings reveal the vital roles of germ cell-specific STYXL1 in CCT-facilitated tubulin folding and sperm flagella development.

Tubulin folding cofactor B regulates microtubule homeostasis through an evolutionarily conserved pathway.

This article is a Commentary on Zhou et al. (2023), 239: 1707–1722.

Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin—with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

Concurrent silencing of TBCE and drug delivery to overcome platinum-based resistance in liver cancer.

Platinum-based chemotherapy resistance is a key factor of poor prognosis and recurrence in hepatocellular carcinoma (HCC). Herein, RNAseq analysis revealed that elevated tubulin folding cofactor E (TBCE) expression is associated with platinum-based chemotherapy resistance. High expression of TBCE contributes to worse prognoses and earlier recurrence among liver cancer patients. Mechanistically, TBCE silencing significantly affects cytoskeleton rearrangement, which in turn increases cisplatin-induced cycle arrest and apoptosis. To develop these findings into potential therapeutic drugs, endosomal pH-responsive nanoparticles (NPs) were developed to simultaneously encapsulate TBCE siRNA and cisplatin (DDP) to reverse this phenomena. NPs (siTBCE+DDP) concurrently silenced TBCE expression, increased cell sensitivity to platinum treatment, and subsequently resulted in superior anti-tumor effects both invitro and invivo in orthotopic and patient-derived xenograft (PDX) models. Taken together, NP-mediated delivery and the co-treatment of siTBCE+DDP proved to be effective in reversing chemotherapy resistance of DDP in multiple tumor models.

The Role of TRiC-enhanced Actin Folding in Leber Congenital Amaurosis.

Mutations in TCP-1 ring complex (TRiC) have been associated with Leber Congenital Amaurosis (LCA). TRiC is involved in protein folding and has 8 essential subunits including CCT5. Herein, we studied the retina of TRiC mutant zebrafish to evaluate the possible role of impaired actin and tubulin folding in LCA. The cct5 retina was histologically studied using Toluidine Blue staining as well as TUNEL, BrdU-labeling, and Phalloidin assays. Retinal organisation was assessed by quantification of the cellularity utilising DAPI. Laminar organization of cct5 retinas was intact. Enhanced apoptosis throughout the cct5 retina was not compensated by higher proliferation rates, leaving the cct5 retina smaller in size. Quantification of retinal layer cellularity demonstrated that specifically the numbers of the amacrine and the retinal ganglion cells were depleted, suggesting that the cct5 retina was not uniformly affected by the reduced actin folding. Whereas the current literature suggests that LCA is predominantly affecting retinal photoreceptor cells and the retinal pigment epithelium, cct5 analyses demonstrated the important role of folding of actin by TRiC, suggesting that cct5 is a useful tool to specifically analyze the role of F-actin filaments in the context of LCA.

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

Snapshots of actin and tubulin folding inside the TRiC chaperonin

The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.

A co-expression network based molecular characterization of genes responsive for Braak stages in Parkinson's disease.

The progression of Parkinson's disease (PD) is defined by six Braak stages. We used transcriptome data from PD patients with Braak stage information to understand underlying molecular mechanisms for the progress of the disease. We created networks of genes with continuously decreased/increased co-expression from control group to Braak 5-6 stages. These networks are significantly associated with PD-related mechanisms such as mitochondrial dysfunction and synaptic signalling among others. Applying weighted gene co-expression network analysis (WGCNA) algorithm to the co-expression networks led to more specific modules enriched with neurodegeneration-related disease pathways, seizure, abnormality of coordination, and hypotonia. Furthermore, we showed that one of the co-expression networks is clustered into three major communities with dedicated molecular functions: (i) tubulin folding pathway, gap junction-related mechanisms, neuronal system; (ii) synaptic vesicle, intracellular vesicle, proteasome complex, PD genes; (iii) energy metabolism, mitochondrial mechanisms, oxidative phosphorylation, TCA cycle, PD genes. The co-expression relations we identified in this study as crucial players in the disease progression cover several known PD-associated genes and genes whose products are known to physically interact with alpha-synuclein protein.

Tubulin-folding cofactor E deficiency is associated with vascular dysfunction and endoplasmatic reticulum stress of vascular smooth muscle cells

Abstract Introduction Endothelial function assessed via flow mediated dilatation (FMD) has shown to predict risk in individuals with established cardiovascular diseases, whereas its predictive value is uncertain in the setting primary prevention. Purpose The aim of the current work was to discover and evaluate novel mediators of vascular dysfunction in the general population and in conditional knock-out transgenic animal models. Methods In order to identify novel targets that were negatively correlated with FMD and investigate their contribution in vascular function, a Genome Wide Association Study (GWAS) of 5,000 participants was performed and subsequently immune cell-, endothelial- and vascular smooth muscle cell (VSMCs)-targeted conditional knockout mouse models were generated and characterized. Results GWAS analysis revealed that single nucleotide polymorphisms (SNPs) in the tubulin folding cofactor E (TBCE) gene were negatively correlated with FMD and TBCE expression in the peripheral blood mononuclear cells (PBMCs). Myelomonocytic cell-targeted TBCE deficiency did not lead to any vascular dysfunction in vivo in the LysM+Cre+/−TBCEfl/fl mice. Endothelial-targeted TBCE deficiency led to an NLR family pyrin domain containing 3 (NLRP3)-dependent activation of the inflammasome in the endothelial cells of Tie2-ERT2Cre+/−TBCEfl/fl mice. Importantly, VSMC-targeted TBCE deficiency was associated with endothelial dysfunction, increased aortic wall thickness and endoplasmatic reticulum (ER) stress-mediated VSMC hyperproliferation in vivo (SMMHC-ERT2Cre+/−TBCEfl/fl), paralleled by calnexin upregulation. Administration of the blood pressure hormone angiotensin II exacerbated the vascular dysfunction and phenotype. Administration of the ER stress modulator tauroursodeoxycholic acid to the SMMHC-ERT2Cre+/−TBCEfl/fl mice reversed vascular dysfunction, paralleled by induction of Raptor/Beclin-1-dependent autophagy both in vitro and in vivo. Conclusion TBCE and tubulin homeostasis in the vascular musculature seem to be novel markers of vascular function and represent a new druggable target for the treatment of ER-stress-mediated vascular dysfunction. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): This work was supported by grants of the German Federal Ministry for Education and Research (BMBF01EO1003 and BMBF01EO1503), the DFG Major Research Instrumentation Programme (DFG INST 371/47-1 FUGG) as well as the Boehringer Ingelheim Foundation. PW received funds from the German Research Foundation in support of his work (DFG WE4361-4-1 and WE 4361/7-1). KS, TM and PW are PIs of the DZHK.

Sanjad Sakati syndrome and sleep-disordered breathing: an undisclosed association.

Sanjad Sakati syndrome (SSS) is a rare autosomal recessive genetic disorder caused by mutation in TBCE (tubulin folding cofactor E) gene. Reported cases were almost exclusively of Middle-Eastern and Arabian children of consanguineous parents. We report the clinical manifestations, outcome, and an observed new association of sleep-disordered breathing (SDB) among children with Sanjad Sakati syndrome. Clinical and routine laboratory data of SSS cases attending Sultan Qaboos University Hospital, Oman, were collected from the electronic patient records or through direct clinic interviews. In-lab polysomnography (PSG) and echocardiography were carried out for all the cases. SDB diagnosis was based on the guidelines of the American Academy of Sleep Medicine. Of 12 patients with SSS, 5 males (42%), all of them (100%) had obstructive sleep apnea (OSA) and 4 of them(33%) had additional significant central apnea and sleep-related hypoventilation. Eight patients (67%) had severe SDB with mean apnea-hypopnea index (AHI) of 26.5 events/h. Age at time of diagnosis with SDB ranged from 2 to 17years with mean of 8.9 [Formula: see text] Two patients had severe pulmonary hypertension as a complication of severe SDB and died from type 2 respiratory failure. Sleep-disordered breathing is prevalent among children with SSS, especially OSA. This is the first study to report SDB in a large cohort of patients with this extremely rare syndrome. The study results encourage the importance of screening affected patients with SSS for sleep-disordered breathing early before developing severe morbidities such as pulmonary hypertension that further compromise their quality of life.

Retraction: Leishmania-Specific Promiscuous Membrane Protein Tubulin Folding Cofactor D Divulges Th1/Th2 Polarization in the Host via ERK-1/2 and p38 MAPK Signaling Cascade.

[This retracts the article DOI: 10.3389/fimmu.2020.00817.].

Colchicine Blocks Tubulin Heterodimer Recycling by Tubulin Cofactors TBCA, TBCB, and TBCE.

Colchicine has been used to treat gout and, more recently, to effectively prevent autoinflammatory diseases and both primary and recurrent episodes of pericarditis. The anti-inflammatory action of colchicine seems to result from irreversible inhibition of tubulin polymerization and microtubule (MT) assembly by binding to the tubulin heterodimer, avoiding the signal transduction required to the activation of the entire NLRP3 inflammasome. Emerging results show that the MT network is a potential regulator of cardiac mechanics. Here, we investigated how colchicine impacts in tubulin folding cofactors TBCA, TBCB, and TBCE activities. We show that TBCA is abundant in mouse heart insoluble protein extracts. Also, a decrease of the TBCA/β-tubulin complex followed by an increase of free TBCA is observed in human cells treated with colchicine. The presence of free TBCA is not observed in cells treated with other anti-mitotic agents such as nocodazole or cold shock, neither after translation inhibition by cycloheximide. In vitro assays show that colchicine inhibits tubulin heterodimer dissociation by TBCE/TBCB, probably by interfering with interactions of TBCE with tubulin dimers, leading to free TBCA. Manipulation of TBCA levels, either by RNAi or overexpression results in decreased levels of tubulin heterodimers. Together, these data strongly suggest that TBCA is mainly receiving β-tubulin from the dissociation of pre-existing heterodimers instead of newly synthesized tubulins. The TBCE/TBCB+TBCA system is crucial for controlling the critical concentration of free tubulin heterodimers and MT dynamics in the cells by recycling the tubulin heterodimers. It is conceivable that colchicine affects tubulin heterodimer recycling through the TBCE/TBCB+TBCA system producing the known benefits in the treatment of pericardium inflammation.

White-Sutton syndrome with hot water epilepsy and coexistence of SHOX gene variations.

The purpose of this study is to reveal the effect on the clinical phenotype of variants detected at family examination of a case of combined pogo transposable element derived with zinc finger domain (POGZ) gene, tubulin folding cofactor E (TBCE) gene, and short stature homeobox (SHOX) gene variation. A Turkish non-consanguineous family consisting of five members was investigated. Whole exome sequence analysis and chromosomal microarray analysis (CMA) were performed for a 2-year-old male patient (the proband) with global developmental delay, hypotonia, dysmorphia, and hot water epilepsy. Targeted sequence and chromosomal microarray analyses were performed for each family member. A heterozygous c.3908_3911delTCTG/p.V1303fs*6 variant was detected in the POGZ gene and a heterozygous c.626T > G(p.L209X) variant in the TBCE gene in the proband. In addition, a gain of 0.1MB was detected in the Xp22.33(602488-733497) × 3/Yp11.32(552488-683497) × 3 region at CMA. The SHOX (312865) gene defined in Online Mendelian Inheritance in Man is located in this region. While the proband's father and brother had heterozygous variations only in the TBCE gene, neither TBCE nor POGZ mutations were detected in the mother or sister. A gain in Xp22.33(419224-883640) × 3 was detected in the mother at CMA. Except for short stature and Madelung deformity, no phenotypical findings were detected in the mother. Other family members were also phenotypically normal. The family screening confirmed that dysmorphic findings and global developmental delay in the proband resulted from the variation in the POGZ gene, while short stature was caused by the gain in the Xp22.33(602488-733497) × 3/Yp11.32(552488-683497) × 3 region. In addition, the pathogenic POGZ gene variation in our patient may be a possible cause of hot water epilepsy. Heterozygous variation in the TBCE gene was clinically insignificant. Hot water epilepsy has not previously been reported in the rare patients with POGZ gene mutation. Additionally, in contrast to the previous literature, the proband exhibited no features of autism. It should also be remembered that posterior fossa abnormalities are frequently seen in these patients. We think that this case and family review involving POGZ and SHOX gene mutations will make a useful contribution to the existing literature.

Tubulin Folding Cofactor D Deficiency: Missing the Diagnosis With Whole Exome Sequencing.

Two siblings with an early onset of a neurodegenerative disease were presented with muscular hypotonia, secondary microcephaly, and severe developmental delay. Seizures were refractory to treatment but could be controlled with a ketogenic diet. Over the course of 5 years, whole exome sequencing (WES) was performed twice in both children. The first time the diagnosis was missed. The next one revealed compound heterozygous mutations in the gene coding for the tubulin folding cofactor D. Technical improvements in WES mandated a new investigation after a few years in children where the diagnosis has not been found.

Short-term Exposure to Ambient Particulate Elements and Epigenome-wide DNA Methylation in Older Men: the Normative Aging Study

Background: Current research suggests that short- and intermediate- term exposure to ambient particulate matter is associated with global and gene-specific DNA methylation (DNAm), but little is known regarding the changes in epigenome-wide caused by particulate elements (PEs). Additionally, the potential pathways remain unknown.Objective: We evaluated short- to intermediate-term associations between ambient PEs and DNAm in epigenome-wide and identified potential biological pathways.Method: We collected whole blood samples from 691 elderly men in the Normative Aging Study (1999-2013) and measured epigenome-wide DNAm with the Illumina Infinium HumanMethylation450 BeadChip. Ambient PEs were measured daily at a fixed monitoring station and 5 different moving averages from short- to intermediate-term (current day to 28-day) were considered. Linear quantile mixed models were used to estimate the associations between 15 elements and DNAm (N=1, 262 visits), adjusting for fine particle mass, cell type, batch effects, age, body mass index, smoking, etc. Pathway analysis was also performed to explore the potential biologic mechanisms.Results: Short- to intermediate-term exposure to PEs was significantly associated with epigenome-wide DNAm, especially for 28-d moving average. We observed different Bonferroni significant CpGs for different PE, most of them related with aluminum, iron, silicon, lead, and, calcium. The pathway analyses suggest that the detrimental effects might be relevant to cell cycle, biosynthetic process, and signal transduction. We also found that there were several common genes significantly associated with calcium, silicon and lead, such as, neurodegenerative disease related gene tubulin folding cofactor D.Conclusions: Ours is the first study that find short- to intermediate-term effects on DNAm in epigenome-wide by ambient PEs, especially elements associated with road dust, oil combustion, and wood burning. Our findings provide insight into potential mechanistic pathways.

An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.