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Hannemann M.,European Neuroscience Institute ENI | Hannemann M.,International Max Planck Research School Molecular Biology | Sasidharan N.,European Neuroscience Institute ENI | Sasidharan N.,International Max Planck Research School Neuroscience | And 10 more authors.
PLoS Genetics | Year: 2012

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. © 2012 Hannemann et al.


Kabatas S.,University of Gottingen | Vreja I.C.,University of Gottingen | Vreja I.C.,International Max Planck Research School Molecular Biology | Saka S.K.,University of Gottingen | And 7 more authors.
Chemical Communications | Year: 2015

Imaging techniques should differentiate between specific signals, from the biomolecules of interest, and non-specific signals, from the background. We present a probe containing 15N and 14N isotopes in approximately equal proportion, for secondary ion mass spectrometry imaging. This probe designed for a precise biomolecule analysis is insensitive to background signals. © 2015 The Royal Society of Chemistry.


Denker A.,European Neuroscience Institute | Denker A.,International Max Planck Research School Molecular Biology | Rizzoli S.O.,European Neuroscience Institute
Frontiers in Synaptic Neuroscience | Year: 2010

During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or "pools". We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are "fixed". Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool. © 2010 Denker and Rizzoli.


Vreja I.C.,University of Gottingen | Vreja I.C.,Center for Nanoscale Microscopy and Molecular Physiology of the Brain | Vreja I.C.,International Max Planck Research School Molecular Biology | Kabatas S.,University of Gottingen | And 12 more authors.
Angewandte Chemie - International Edition | Year: 2015

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added 19F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The 19F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms. SPILLing the beans: a method of labeling specific proteins for secondary-ion mass spectrometry (SIMS), termed SPILL (specific protein isotopic and fluorescence labeling) is developed which involves unnatural amino acid incorporation and click reaction with a fluorescent probe enriched in 19F (see scheme). The applicability of this method extends from cell culture systems to invertebrate model organisms. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.


Saka S.,European Neuroscience Institute | Saka S.,International Max Planck Research School Molecular Biology | Rizzoli S.O.,European Neuroscience Institute
BioEssays | Year: 2012

The use of super-resolution imaging techniques in cell biology has yielded a wealth of information regarding cellular elements and processes that were invisible to conventional imaging. Focusing on images obtained by stimulated emission depletion (STED) microscopy, we discuss how the new high-resolution data influence the ways in which we use and interpret images in cell biology. Super-resolution images have lent support to some of our current hypotheses. But, more significantly, they have revealed unexpectedly complex processes that cannot be accounted for by the simpler models based on diffraction-limited imaging. The super-resolution imaging data challenge cell biologists to change their theoretical framework, by including, for instance, interpretations that describe multiple functions, functional errors or lack of function for cellular elements. In this context, we argue that descriptive research using super-resolution microscopy is now as necessary as hypothesis-driven research. © 2012 WILEY Periodicals, Inc..

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