A neurodevelopmental disorder associated with a loss-of-function missense mutation in RAB35.

Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.

DENND6A links Arl8b to a Rab34/RILP/dynein complex, regulating lysosomal positioning and autophagy.

Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.

Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence.

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Coiled-coil-helix-coiled-coil-helix domain containing protein 10 (CHCHD10) for use in Western Blot, immunoprecipitation and immunofluorescence.

CHCHD10 is a mitochondrial protein, implicated in the regulation of mitochondrial morphology and cristae structure, as well as the maintenance of mitochondrial DNA integrity. Recently discovered to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in its mutant form, the scientific community would benefit from the availability of validated anti-CHCHD10 antibodies. In this study, we characterized four CHCHD10 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. As this study highlights high-performing antibodies for CHCHD10, we encourage readers to use it as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Profilin-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence.

Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance in Western blot, immunoprecipitation, and immunofluorescence, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Charged multivesicular body protein 2b for use in Western Blot, immunoprecipitation and immunofluorescence.

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii) yet, ~50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

DENND2B activates Rab35 at the intercellular bridge, regulating cytokinetic abscission and tetraploidy.

Cytokinesis relies on membrane trafficking pathways regulated by Rabs and guanine nucleotide exchange factors (GEFs). During cytokinesis, the intercellular cytokinetic bridge (ICB) connecting daughter cells undergoes abscission, which requires actin depolymerization. Rab35 recruits MICAL1 to oxidize and depolymerize actin filaments. We show that DENND2B, a protein linked to cancer and congenital disorders, functions as a Rab35 GEF, recruiting and activating Rab35 at the ICB. DENND2B's N-terminal region also interacts with an active form of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission, leading to multinucleated cells and filamentous actin (F-actin) accumulation at the ICB, impairing recruitment of ESCRT-III at the abscission site. Additionally, F-actin accumulation triggers the formation of a chromatin bridge, activating the NoCut/abscission checkpoint, and DENND2B knockdown activates Aurora B kinase, a hallmark of checkpoint activation. Thus, our study identifies DENND2B as a crucial player in cytokinetic abscission.

The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community.  In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence.

Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for TDP-43 for use in Western Blot, immunoprecipitation and immunofluorescence.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein playing a critical role in the regulation of transcription, splicing and RNA stability. Mutations in TARDBP leading to aggregation, are suspected to be a characteristic feature of various neurogenerative diseases. The lack of well-characterized anti- TDP-43 antibodies acts as a barrier to establish reproducible TDP-43 research. In this study, we characterized eighteen TDP-43 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Visualization of α-synuclein trafficking via nanogold labeling and electron microscopy.

There is conflicting evidence regarding the mechanisms of α-synuclein internalization, and its trafficking itinerary following cellular entry remains largely unknown. To examine these issues, we describe steps for coupling α-synuclein preformed fibrils (PFFs) to nanogold beads and their subsequent characterization by electron microscopy (EM). Then we describe the uptake of conjugated PFFs by U2OS cells plated on Permanox 8-well chamber slides. This process eliminates the reliance on antibody specificity and the need to employ complex immunoEM staining protocols. For complete details on the use and execution of this protocol, please refer to Bayati et al. (2022).1.

A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence.

Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson's disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence.

A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence.

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson's Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence.

TBK1 is a serine-threonine protein kinase that has been linked to a number of diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for TBK1 for use in immunoblot, immunofluorescence and immunoprecipitation, using an isogeneic knock-out cell line as a control. We identify antibodies that appear specific for all three applications but invite the readers to interpret the present findings based on their own scientific expertise and use this report as a guide to select the most appropriate antibody for their specific needs.

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of ß-Coronaviruses.

From a designed library of indolyl pyrimidinamines, we identified a highly potent and cell-active chemical probe (17) that inhibits phosphatidylinositol-3-phosphate 5-kinase (PIKfyve). Comprehensive evaluation of inhibitor selectivity confirmed that this PIKfyve probe demonstrates excellent kinome-wide selectivity. A structurally related indolyl pyrimidinamine (30) was characterized as a negative control that lacks PIKfyve inhibitory activity and exhibits exquisite selectivity when profiled broadly. Chemical probe 17 disrupts multiple phases of the lifecycle of ß-coronaviruses: viral replication and viral entry. The diverse antiviral roles of PIKfyve have not been previously probed comprehensively in a single study or using the same compound set. Our scaffold is a distinct chemotype that lacks the canonical morpholine hinge-binder of classical lipid kinase inhibitors and has a non-overlapping kinase off-target profile with known PIKfyve inhibitors. Our chemical probe set can be used by the community to further characterize the role of PIKfyve in virology.