Biology subjects

Winkenbach, L. P.

Publications and source records attributed to Winkenbach, L. P..

4 recordsLinked to original sources

bioRxiv2026

Differential mRNA localization of karyopherin-β2 homologs in C. elegans and humans

In Caenorhabditis elegans embryos, the nuclear transport receptor IMB-2 (Importin Beta Family-2, a karyopherin {beta}2) preferentially localizes to the nuclear envelope along with its encoding mRNA. This suggests that imb-2 mRNA is locally translated at the nuclear envelope. To test whether imb-2s two putative human orthologs, Transportin 1 (TNPO1) and Transportin 2 (TNPO2), exhibited similar mRNA localization and local translation, we performed smiFISH and microscopy in U2OS, HeLa, and human pluripotent stem cells. Neither human TNPO1 nor TNPO2 mRNA localized to the nuclear envelope in any tested human cell type. However, the human TNPO1 protein and the C. elegans IMB-2 protein both localized to the nucleus and its periphery. This suggests that despite their shared functional roles and high amino acid sequence identities (52% and 51%, respectively), these karyopherins differed in their translational dynamics.

genetics↗

bioRxiv2022

Translation of the ERM-1 membrane-binding domain directs erm-1 mRNA localization to the plasma membrane in the C. elegans embryo

mRNA localization and transport are integral in regulating gene expression. In Caenorhabditis elegans embryos, the maternally inherited mRNA erm-1 (Ezrin/Radixin/Moesin) concentrates in anterior blastomeres. erm-1 mRNA localizes within those blastomeres to the plasma membrane where the essential ERM-1 protein, a membrane-actin linker, is also found. We demonstrate that the localization of erm-1 mRNA to the plasma membrane is translation-dependent and requires its encoded N-terminal membrane-binding (FERM) domain. By perturbing translation through multiple methods, we found erm-1 mRNA localization at the plasma membrane was maintained only if the nascent peptide remained in complex with the translating mRNA. Indeed, recoding the erm-1 mRNA coding sequence while preserving the encoded amino acid sequence did not disrupt erm-1 mRNA localization, corroborating that the information directing mRNA localization resides within its membrane-binding protein domain. A smiFISH screen of 17 genes encoding similar membrane-binding domains identified three plasma membrane localized mRNAs in the early embryo. Nine additional transcripts showed apparent membrane localization later in development. These findings point to a translation-dependent pathway for localization of mRNAs encoding membrane-associated proteins. SUMMARY STATEMENTIn C. elegans, erm-1 mRNA localization to plasma membranes requires translation of the ERM-1 membrane-binding domain

developmental biology↗

bioRxiv2021

Improved methods for protein and single-molecule RNA detection in C. elegans embryos

Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule Fluorescence In Situ Hybridization (smFISH) and Immunofluorescence (IF) visualize the abundance and localization of mRNAs and proteins, respectively, allowing researchers to elucidate the localization, dynamics, and functions of many genes. Here, we describe several improvements and optimizations to existing IF and smFISH approaches specifically for use in C. elegans embryos. We present 1) optimized fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility, 2) a streamlined, in-tube approach that negates freeze-cracking, 3) the smiFISH (single molecule inexpensive FISH) adaptation that reduces cost, 4) an assessment of optimal anti-fade products, and 5) straightforward quantification and data analysis methods. Most importantly, published IF and smFISH protocols have predominantly been mutually exclusive, preventing exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we present methods to combine IF and smFISH protocols in C. elegans embryos including an efficient method harnessing nanobodies. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol.

cell biology↗

bioRxiv2020

mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage

Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription. This presents an opportunity to study mechanisms of post-transcriptional regulatory control. In seeking the mechanisms behind this patterning, we discovered that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage), and P-bodies (associated with RNA processing). Transcripts differed in their dependence on 3UTRs and RNA Binding Proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling these, we untangled a long-standing question: Are mRNAs directed to P granules for translational repression or do they accumulate there as a downstream step? We found translational repression preceded P granule localization and could occur independent of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. Overall, we show transcripts important for germline development are directed to P granules by translational repression, and this, in turn, directs their accumulation in the progenitor germ lineage where their repression can ultimately be relieved. SummaryMaternally loaded mRNAs localize non-homogeneously within C. elegans early embryos correlating with their translational status and lineage-specific fates.

developmental biology↗