Biology subjects

Cameron, C. J.

Publications and source records attributed to Cameron, C. J..

8 recordsLinked to original sources

bioRxiv2024

F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth

Neurite initiation from newly born neurons is a critical step in neuronal differentiation and migration. Neuronal migration in the developing cortex is accompanied by dynamic extension and retraction of neurites as neurons progress through bipolar and multipolar states. However, there is a relative lack of understanding regarding how the dynamic extension and retraction of neurites is regulated during neuronal migration. In recent work we have shown that CIP4, a member of the F-BAR family of membrane bending proteins, inhibits cortical neurite formation in culture, while family member FBP17 induces premature neurite outgrowth. These results beg the question of how CIP4 and FBP17 function in radial neuron migration and differentiation in vivo, including the timing and manner of neurite extension and retraction. Indeed, the regulation of neurite outgrowth is essential for the transitions between bipolar and multipolar states during radial migration. To examine the effects of modulating expression of CIP4 and FBP17 in vivo, we used in utero electroporation, in combination with our published Double UP technique, to compare knockdown or overexpression cells with control cells within the same mouse tissue of either sex. We show that either knockdown or overexpression of CIP4 and FBP17 results in the marked disruption of radial neuron migration by modulating neuronal morphology and neurite outgrowth, consistent with our findings in culture. Our results demonstrate that the F-BAR proteins CIP4 and FBP17 are essential for proper radial migration in the developing cortex and thus play a key role in cortical development. SIGNIFICANCE STATEMENTDuring embryonic development, radial migration of newly born cortical neurons is a complex process that underlies the proper formation of the neocortex, the outermost layers of neurons in the brain. Disruptions in radial migration results in profound effects on cognitive function and can lead to devastating developmental disabilities. To better understand this critical process in brain development we examined two members of the F-BAR family of membrane bending proteins, CIP4 and FBP17, which are present in the developing brain. We demonstrate that intracellular concentrations of these proteins must be tightly regulated. Increasing or decreasing levels of either protein has profound effects on neuronal morphology and proper radial migration, suggesting they are key players in cortical development.

neuroscience↗

bioRxiv2023

REPIC -- an ensemble learning methodology for cryo-EM particle picking

Cryo-EM (cryogenic electron microscopy) particle identification from micrographs (i.e., picking) is challenging due to the low signal-to-noise ratio and lack of ground truth for particle locations. Moreover, current computational methods ("pickers") identify different particle sets, complicating the selection of the best-suited picker for a protein of interest. Here, we present REPIC, an ensemble learning methodology that uses multiple pickers to find consensus particles. REPIC identifies consensus particles by framing its task as a graph problem and using integer linear programming to select particles. REPIC picks high-quality particles when the best picker is not known a priori and for known difficult-to-pick particles (e.g., TRPV1). Reconstructions using consensus particles achieve resolutions comparable to those from particles picked by experts, without the need for downstream particle filtering. Overall, our results show REPIC requires minimal (often no) manual picking and significantly reduces the burden on cryo-EM users for picker selection and particle picking. Availabilityhttps://github.com/ccameron/REPIC

bioinformatics↗

bioRxiv2021

Nodal modulator is required to sustain endoplasmic reticulum morphology

Nodal Modulator (NOMO) is a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We identified NOMO1 in a proteomics approach aimed at the identification of proteins that support the structural integrity of the endoplasmic reticulum (ER). Overexpression of NOMO1 imposes a sheet morphology on the ER, while depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network. These structures are positive for the autophagy marker LAMP1, and LC3 is profoundly upregulated upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a dimeric state that is mediated by the cytosolic tail domain, with each monomer featuring a "beads on a string" structure likely representing bacterial Ig-like folds. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.

cell biology↗

bioRxiv2020

SACSANN: identifying sequence-based determinants of chromosomal compartments

Genomic organization is critical for proper gene regulation and based on a hierarchical model, where chromosomes are segmented into megabase-sized, cell-type-specific transcriptionally active (A) and inactive (B) compartments. Here, we describe SACSANN, a machine learning pipeline consisting of stacked artificial neural networks that predicts compartment annotation solely from genomic sequence-based features such as predicted transcription factor binding sites and transposable elements. SACSANN provides accurate and cell-type specific compartment predictions, while identifying key genomic sequence determinants that associate with A/B compartments. Models are shown to be largely transferable across analogous human and mouse cell types. By enabling the study of chromosome compartmentalization in species for which no Hi-C data is available, SACSANN paves the way toward the study of 3D genome evolution. SACSANN is publicly available on GitHub: https://github.com/BlanchetteLab/SACSANN

bioinformatics↗

bioRxiv2019

LESSeq: Local event-based analysis of alternative splicing using RNA-Seq data

Alternative splicing, which can be observed genome-wide by RNA-Seq, is important in cellular development and evolution. Comparative RNA-Seq experiments between different cellular conditions allow alternative splicing signatures to be detected. However, inferring alternative splicing signatures from short-read technology is unreliable and still presents many challenges before biologically significant signatures may be identified. To enable the robust discovery of differential alternative splicing, we developed the Local Event-based analysis of alternative Splicing using RNA-Seq (LESSeq) pipeline. LESSeq utilizes information of local splicing events (i.e., the partial structures in genes where transcript-splicing patterns diverge) to identify unambiguous alternative splicing. In addition, LESSeq quantifies the abundance of these alternative events using Maximum Likelihood Estimation (MLE) and provides their significance between different cellular conditions. The utility of LESSeq is demonstrated through two case studies relevant to human variation and evolution. Using an RNA-Seq data set of lymphoblastoid cell lines in two human populations, we examined within-species variation and discovered population-differential alternative splicing events. With an RNA-Seq data set of several tissues in human and rhesus macaque, we studied cross-species variation and identified lineage-differential alternative splicing events. LESSeq is implemented in C++ and R, and made publicly available on GitHub at: https://github.com/gersteinlab/LESSeq

bioinformatics↗

bioRxiv2019

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.

genomics↗

bioRxiv2018

Estimating DNA-DNA interaction frequency from Hi-C data at restriction-fragment resolution

Hi-C is a popular technique to map three-dimensional chromosome conformation by capturing the frequency of physical contacts between pairs of genomic regions in cell populations. Although the resolution of Hi-C data is in principle only limited by the size of restriction fragments (300 bp - 4 kb), stochastic noise caused by the limited sequencing coverage forces researchers to artificially reduce the resolution of Hi-C matrices by binning the genome into 5-100 kb regions, resulting in a loss of information and biological interpretability. Here, we present the Hi-C Interaction Frequency Inference (HIFI) algorithms, a family of computational approaches that takes advantage of dependencies between neighboring restriction fragments to estimate restriction-fragment resolution interaction frequency matrices from Hi-C data. HIFI is shown to be superior to existing fixed-binning and state-of-the-art approaches via cross-validation experiments on Hi-C data and comparisons to 5C data. It also greatly improves the delineation of enhancer-promoter contacts. Finally, the high resolution afforded by HIFI reveals a new role for active regulatory regions in structuring topologically associating domains (TADs) and subTADs. By operating upstream of many Hi-C data analysis tools (e.g., normalization tools, as well as loop, TAD, and compartment predictors), HIFI will be easily inserted into a number of Hi-C data analysis pipelines, enabling a variety of high-resolution genomic organization analyses.\n\nAvailabilitygithub.com/BlanchetteLab/HIFI

bioinformatics↗

bioRxiv2018

Efficient Homology Directed Repair by Cas9:DNA Localization and Cationic Polymeric Transfection in Mammalian Cells

Homology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.\n\nSummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

biochemistry↗