Fluorescence Time-lapse Imaging of the Complete &lt;em&gt;S. venezuelae&lt;/em&gt; Life Cycle Using a Microfluidic Device

Schlimpert, Susan; Flärdh, Klas; Buttner, Mark J.

doi:10.3791/53863

Cited by 40 publications

(68 citation statements)

References 31 publications

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“…The coverslip was then moved on top of a thin agarose pad. Fluorescent time-lapse imaging was essentially performed as described in Schlimpert et al (29). Spores were loaded into B04A microfluidic plates (ONIX; CellASIC) and allowed to germinate and grow by perfusing MYM for 3 h. Sporulation was induced by switching the media flow channel and incubating the growing hyphae in spent MYM, which was derived by filter-sterilizing the growth medium from a sporulating culture.…”

Section: Methodsmentioning

confidence: 99%

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Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Schlimpert

Wasserström

Chandra

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.he active organization and remodeling of cellular membranes is a fundamental process for all organisms. Many of these remodeling events involve the reshaping, fission, or fusion of lipid bilayers to generate new organelles, release transport vesicles, or stabilize specific membrane structures. In eukaryotes, key cellular processes, such as the fission and fusion of mitochondria, the division of chloroplasts, endocytosis, and viral resistance, are mediated by members of the dynamin superfamily (1). Dynamins and dynamin-like proteins are mechanochemical GTPases that polymerize into helical scaffolds at the surface of membranes. GTP hydrolysis is coupled to a radical conformational change in the protein structure that forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements, probably via a hemifusion intermediate (2, 3).Dynamin-like proteins are found in many bacterial species, yet the precise roles of these proteins are still largely unknown. They share a conserved domain architecture with the canonical human Dynamin 1, including the N-terminal GTPase domain, a neck domain involved in dynamin dimerization, and a trunk domain involved in stimulation of GTPase activity (4, 5). In contrast, bacterial dynamins lack the pleckstrin homology motif and proline-rich sequences found in classical dynamins and contain instead o...

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Section: Methodsmentioning

confidence: 99%

“…During this process, helical FtsZ filaments tumble along the hypha and then coalesce into long ladders of regularly spaced Z-rings (28,29). However, the molecular mechanisms that control the stability and functionality of these multiple division-competent Z-rings are unknown.…”

mentioning

confidence: 99%

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Schlimpert

Wasserström

Chandra

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…We imaged the sporulation of Streptomyces venezuelae hyphae (17) by exposing them to the spent media of a sporulated culture either in microfluidic chambers (see Movie S2 and Fig. S4 in the supplemental material) or on agarose pads (Fig.…”

Section: Observationmentioning

confidence: 99%

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria

Zhou

Halladin

Theriot

2016

mBio

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Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria. In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae.

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“…Studies of surface-grown cultures are bedevilled by developmental asynchrony and heterogeneity, which are particularly problematic for high-resolution biochemical analysis, including “omic” approaches to the definition of the regulons controlled by developmental regulatory genes. These problems have been circumvented by the adoption of S. venezuelae , which grows very rapidly and undergoes comprehensive and near-synchronous differentiation in liquid culture, including under coverslips 68 . In a first illustration of the value of this system, a detailed time-lapse study has shown that the ParA and ParB chromosome segregation proteins play an important coordinating role in the transition from ongoing aerial growth to growth cessation and the onset of sporulation septation, and even have opposing influences on the rate of tip extension and eventual length of pre-sporulation hyphae 69 .…”

Section: New Perspectives In Growth and Development Of Streptomycesmentioning

confidence: 99%

“…In a first illustration of the value of this system, a detailed time-lapse study has shown that the ParA and ParB chromosome segregation proteins play an important coordinating role in the transition from ongoing aerial growth to growth cessation and the onset of sporulation septation, and even have opposing influences on the rate of tip extension and eventual length of pre-sporulation hyphae 69 . Further advances are promised by the development of a microfluidics system that allows continuous direct observation of fluorescently marked key proteins through the entire developmental cycle 68 .…”

Section: New Perspectives In Growth and Development Of Streptomycesmentioning

confidence: 99%

Recent advances in understanding Streptomyces

2016

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About 2,500 papers dated 2014–2016 were recovered by searching the PubMed database for Streptomyces, which are the richest known source of antibiotics. This review integrates around 100 of these papers in sections dealing with evolution, ecology, pathogenicity, growth and development, stress responses and secondary metabolism, gene expression, and technical advances. Genomic approaches have greatly accelerated progress. For example, it has been definitively shown that interspecies recombination of conserved genes has occurred during evolution, in addition to exchanges of some of the tens of thousands of non-conserved accessory genes. The closeness of the association of Streptomyces with plants, fungi, and insects has become clear and is reflected in the importance of regulators of cellulose and chitin utilisation in overall Streptomyces biology. Interestingly, endogenous cellulose-like glycans are also proving important in hyphal growth and in the clumping that affects industrial fermentations. Nucleotide secondary messengers, including cyclic di-GMP, have been shown to provide key input into developmental processes such as germination and reproductive growth, while late morphological changes during sporulation involve control by phosphorylation. The discovery that nitric oxide is produced endogenously puts a new face on speculative models in which regulatory Wbl proteins (peculiar to actinobacteria) respond to nitric oxide produced in stressful physiological transitions. Some dramatic insights have come from a new model system for Streptomyces developmental biology, Streptomyces venezuelae, including molecular evidence of very close interplay in each of two pairs of regulatory proteins. An extra dimension has been added to the many complexities of the regulation of secondary metabolism by findings of regulatory crosstalk within and between pathways, and even between species, mediated by end products. Among many outcomes from the application of chromosome immunoprecipitation sequencing (ChIP-seq) analysis and other methods based on “next-generation sequencing” has been the finding that 21% of Streptomyces mRNA species lack leader sequences and conventional ribosome binding sites. Further technical advances now emerging should lead to continued acceleration of knowledge, and more effective exploitation, of these astonishing and critically important organisms.

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Fluorescence Time-lapse Imaging of the Complete <em>S. venezuelae</em> Life Cycle Using a Microfluidic Device

Cited by 40 publications

References 31 publications

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria

Recent advances in understanding Streptomyces

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