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4 results on '"Frank Wippich"'

1. Nutritional stress-induced regulation of microtubule organization and mRNP transport by HDAC1 controlled α-tubulin acetylation

Author

Frank Wippich, Vaishali, Marco L. Hennrich, and Anne Ephrussi

Subjects
Biology (General), QH301-705.5
Abstract

Abstract In response to nutritional stress, microtubules in cells of the Drosophila female germline are depleted from the cytoplasm and accumulate cortically. This triggers aggregation of mRNPs into large processing bodies (P-bodies) and oogenesis arrest. Here, we show that hyperacetylation of α-tubulin at lysine 40 (K40) alters microtubule dynamics and P-body formation. We found that depletion of histone deacetylase 1 (HDAC1) by RNAi phenocopies the nutritional stress response, causing α-tubulin hyperacetylation and accumulation of maternally deposited mRNPs in P-bodies. Through in vitro and in vivo studies, we identify HDAC1 as a direct regulator of α-tubulin K40 acetylation status. In well-fed flies, HDAC1 maintains low levels of α-tubulin acetylation, enabling the microtubule dynamics required for mRNP transport. Using quantitative phosphoproteomics we identify nutritional stress-induced changes in protein phosphorylation that act upstream of α-tubulin acetylation, including phosphorylation of HDAC1 at S391, which reduces its ability to deacetylate α-tubulin. These results reveal that Drosophila HDAC1 senses and relays the nutritional status, which regulates germline development through modulation of cytoskeleton dynamics.

Published
2023
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2. Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture

Author

Imre Gáspár, Frank Wippich, and Anne Ephrussi

Subjects
Biology (General), QH301-705.5
Abstract

Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

Published
2018
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3. Transcript specific mRNP capture from Drosophila egg-chambers for proteomic analysis

Author

Frank Wippich and Anne Ephrussi

Subjects
Proteomics, Messenger RNA, Oligonucleotide, Quantitative proteomics, RNA-binding protein, Biology, General Biochemistry, Genetics and Molecular Biology, Cell biology, Gene Expression Regulation, Ribonucleoproteins, Biotinylation, Gene expression, Animals, Drosophila, Molecular Biology, Post-transcriptional regulation, Protein Binding, Ribonucleoprotein
Abstract

mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes (RNPs) from Drosophila egg-chambers. The method, which is based on in-solution hybridization of short biotinylated antisense DNA oligonucleotide probes to multiple segments of a transcript of interest allows unbiased identification of associated proteins by quantitative proteomics.

Published
2020
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4. Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2

Author

Frank Wippich, Claudia A. O. Stuermer, Friederike A. Jaeger, Helmut Plattner, Georg Luxenhofer, Alexander Reuter, and Matthias F. Langhorst

Subjects
Scaffold protein, Histology, Prions, Golgi Apparatus, Nerve Tissue Proteins, Membrane trafficking, Biology, Endocytosis, Microtubules, PC12 Cells, Clathrin, Exocytosis, Pathology and Forensic Medicine, Jurkat Cells, symbols.namesake, Membrane Microdomains, Epidermal growth factor, ddc:570, Animals, Humans, Vesicle, Reggie/flotillin, Lipid microdomain, Membrane Proteins, Cell Biology, General Medicine, Golgi apparatus, Protein Structure, Tertiary, Rats, Cell biology, Endocytic cycling, Protein Transport, Cholesterol, Microscopy, Fluorescence, Mutation, biology.protein, symbols, HeLa Cells, TIRF microscopy
Abstract

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.

Published
2008
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