TonB dependent transporters (TBDT) are an essential protein family in bacteria involved in the uptake of a broad variety of molecules such as siderophore-chelated iron, which was the first described substrate. Meanwhile it is known that TBDTs are involved in the uptake of many metals, sugars and polypeptides. The action of TBDTs is regulated and energized by the plasma membrane anchored TonB, which is charged by a proton pump. The number of the genes coding for TBDTs varies in different species, which might reflect environmental adaptations or evolutionary variations of the system. For example, in the cyanobacterium Anabaena sp. PCC 7120 the large number of 22 genes coding for TBDTs has been identified and the expression of these genes has been explored in the absence of iron or copper as well as under nitrogen starvation. We describe the analysis of the expression of the TBDT genes and the according cytoplasmic-membrane localized components; the latter appear to have a lower degree of complexity in Anabaena sp. PCC 7120. This analysis unravels that the response is not sole dependent on the metal supply, but also on cell culture densities. In addition, we present a large group of FhuA-like genes which is expressed highest under standard conditions suggesting a function distinct from iron or copper transport. The genes are clustered according to the expression profile and the consequences for our understanding of the transport systems in Anabaena sp. PCC 7120 are discussed.
Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short-and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow. Video LinkThe video component of this article can be found at
Cephalization is a major innovation of animal evolution and implies a synchronization of nervous system, mouth, and foregut polarization to align alimentary tract and sensomotoric system for effective foraging. However, the underlying integration of morphogenetic programs is poorly understood. Here, we show that invagination of neuroectoderm through de novo polarization and apical constriction creates the mouth opening in the Caenorhabditis elegans embryo. Simultaneously, all 18 juxta-oral sensory organ dendritic tips become symmetrically positioned around the mouth: While the two bilaterally symmetric amphid sensilla endings are towed to the mouth opening, labial and cephalic sensilla become positioned independently. Dendrite towing is enabled by the pre-polarized sensory amphid pores intercalating into the leading edge of the anteriorly migrating epidermal sheet, while apical constriction-mediated cell–cell re-arrangements mediate positioning of all other sensory organs. These two processes can be separated by gradual inactivation of the 26S proteasome activator, RPN-6.1. Moreover, RPN-6.1 also shows a dose-dependent requirement for maintenance of coordinated apical polarization of other organs with apical lumen, the pharynx, and the intestine. Thus, our data unveil integration of morphogenetic programs during the coordination of alimentary tract and sensory organ formation and suggest that this process requires tight control of ubiquitin-dependent protein degradation.
Summary: Many developmental processes are inherently robust due to network organization of the participating factors and functional redundancy. The heterogeneity of the factors involved and their connectivity puts these processes at risk of abrupt system collapse under stress. The polarization of the one-cell C. elegans embryo constitutes such an inherently robust process with functional redundancy. However, how polarization is affected by acute stress has not been thoroughly investigated. Here, we report that heat shock (348C, 1 h) triggers a highly reproducible loss of the anterior and collapse of the posterior polarity domains. Temperature-dependent loss of cortical nonmuscle myosin II drastically reduces cortical tension and leads to internalization of large plasma membrane domains including the membrane-associated polarity factor PAR-2. After internalization, plasma membrane vesicles and associated factors cluster around centrosomes and are thereby withdrawn from the polarization process. Transient formation of the posterior polarity domain suggests that microtubule-induced self-organization of this domain is not compromised after heat shock. Hence, our data uncover that the polarization system undergoes a temperature-dependent collapse under acute stress. genesis 54:220-228, 2016. V C 2016Wiley Periodicals, Inc.
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