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Utrecht, Netherlands

Herpers B.,Cell Microscopy Center
Nature protocols | Year: 2010

This protocol describes the combination of in situ hybridization (ISH) with cryo-immunolabeling methods to allow the simultaneous detection at the ultrastructural level of mRNAs and proteins. The procedure consists of five steps and takes 4-5 d: (i) acquisition of ultrathin frozen sections of chemically fixed tissues or cells; (ii) hybridization of the sections with digoxigenin (DIG) or biotin-labeled RNA probes; (iii) detection of the bound probe with antibodies and protein A-gold (PAG); (iv) labeling of proteins of interest (optional); and (v) visualization by transmission electron microscopy (immuno-electron microscopy (IEM)). This technique allows the simultaneous detection of endogenous/overexpressed/injected RNAs and proteins while preserving the cell ultrastructure. The protocol is also suitable for mRNA detection on semi-thin frozen sections in combination with immunofluorescence. The localization of targeted transcripts, such as gurken and oskar mRNA in the Drosophila oocyte, and of structural elements and proteins that mediate their localization have been revealed using this technique.


Tersteeg C.,Laboratory of Clinical Chemistry and Haematology | Tersteeg C.,Laboratory of Experimental Cardiology | Heijnen H.F.,Laboratory of Clinical Chemistry and Haematology | Heijnen H.F.,Cell Microscopy Center | And 10 more authors.
Circulation Research | Year: 2014

RATIONALE:: Platelets are the most important cells in the primary prevention of blood loss after injury. In addition, platelets are at the interface between circulating leukocytes and the (sub)endothelium regulating inflammatory responses. OBJECTIVE:: Our aim was to study the dynamic process that leads to the formation of procoagulant and proinflammatory platelets under physiological flow. METHODS AND RESULTS:: In the present study, we describe the formation of extremely long, negatively charged membrane strands that emerge from platelets adhered under flow. These flow-induced protrusions (FLIPRs) are formed in vitro on different physiological substrates and are also detected in vivo in a mouse carotid injury model. FLIPRs are formed downstream the adherent and activated platelets and reach lengths of 250 μm. FLIPR formation is shear-dependent and requires cyclophilin D, calpain, and Rac1 activation. It is accompanied by a disassembly of the F-actin and microtubule organization. Monocytes and neutrophils roll over FLIPRs in a P-selectin/P-selectin glycoprotein ligand-1-dependent manner, retrieving fragments of FLIPRs as microparticles on their surface. Consequently, monocytes and neutrophils become activated, as demonstrated by increased CD11b expression and L-selectin shedding. CONCLUSIONS:: The formation of long platelet membrane extensions, such as the ones presented in our flow model, may pave the way to generate an increased membrane surface for interaction with monocytes and neutrophils. Our study provides a mechanistic model for platelet membrane transfer and the generation of monocyte/neutrophil-microparticle complexes. We propose that the formation of FLIPRs in vivo contributes to the well-established proinflammatory function of platelets and platelet-derived microparticles. © 2014 American Heart Association, Inc.


Kondylis V.,Cell Microscopy Center | Kondylis V.,University of Cologne | Tang Y.,Cell Microscopy Center | Tang Y.,Tianjin Institute of Urological Surgery | And 5 more authors.
PLoS ONE | Year: 2011

Background: In Drosophila, the early secretory apparatus comprises discrete paired Golgi stacks in close proximity to exit sites from the endoplasmic reticulum (tER sites), thus forming tER-Golgi units. Although many components involved in secretion have been identified, the structural components sustaining its organisation are less known. Here we set out to identify novel ER resident proteins involved in the of tER-Golgi unit organisation. Results: To do so, we designed a novel screening strategy combining a bioinformatics pre-selection with an RNAi screen. We first selected 156 proteins exhibiting known or related ER retention/retrieval signals from a list of proteins predicted to have a signal sequence. We then performed a microscopy-based primary and confirmation RNAi screen in Drosophila S2 cells directly scoring the organisation of the tER-Golgi units. We identified 49 hits, most of which leading to an increased number of smaller tER-Golgi units (MG for "more and smaller Golgi") upon depletion. 16 of them were validated and characterised, showing that this phenotype was not due to an inhibition in secretion, a block in G2, or ER stress. Interestingly, the MG phenotype was often accompanied by an increase in the cell volume. Out of 6 proteins, 4 were localised to the ER. Conclusions: This work has identified novel proteins involved in the organisation of the Drosophila early secretory pathway. It contributes to the effort of assigning protein functions to gene annotation in the secretory pathway, and analysis of the MG hits revealed an enrichment of ER proteins. These results suggest a link between ER localisation, aspects of cell metabolism and tER-Golgi structural organisation. © 2011 Kondylis et al.


Zacharogianni M.,Cell Microscopy Center | Zacharogianni M.,Hubrecht Institute for Developmental Biology and Stem Cell Research | Kondylis V.,Cell Microscopy Center | Kondylis V.,University of Cologne | And 10 more authors.
EMBO Journal | Year: 2011

RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic-Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. We found that ERK7 negatively regulates secretion in response to serum and amino-acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C-terminus of Sec16, a key ER exit site component. This post-translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. We found that ER exit site disassembly upon starvation is TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth. © 2011 European Molecular Biology Organization.


Hoogenraad C.C.,Erasmus Medical Center | Popa I.,University Utrecht | Futai K.,Massachusetts Institute of Technology | Sanchez-Martinez E.,University Utrecht | And 15 more authors.
PLoS Biology | Year: 2010

The endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1 (GRASP-1) as a neuron-specific effector of Rab4 and key component of the molecular machinery that coordinates recycling endosome maturation in dendrites. We show that GRASP-1 is necessary for AMPA receptor recycling, maintenance of spine morphology, and synaptic plasticity. At the molecular level, GRASP-1 segregates Rab4 from EEA1/Neep21/Rab5-positive early endosomal membranes and coordinates the coupling to Rab11-labelled recycling endosomes by interacting with the endosomal SNARE syntaxin 13. We propose that GRASP-1 connects early and late recycling endosomal compartments by forming a molecular bridge between Rab-specific membrane domains and the endosomal SNARE machinery. The data uncover a new mechanism to achieve specificity and directionality in neuronal membrane receptor trafficking. © 2010 Hoogenraad et al.

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