scholarly journals In vivo visualization of type II plasmid segregation: bacterial actin filaments pushing plasmids

2007 ◽  
Vol 179 (5) ◽  
pp. 1059-1066 ◽  
Author(s):  
Christopher S. Campbell ◽  
R. Dyche Mullins
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Fluorescence Microscopy ◽  
Actin Filaments ◽  
Diffusion Constant ◽  
Time Lapse ◽  
Search Space ◽  
Type Ii ◽  
Plasmid Segregation

Type II par operons harness polymerization of the dynamically unstable actin-like protein ParM to segregate low-copy plasmids in rod-shaped bacteria. In this study, we use time-lapse fluorescence microscopy to follow plasmid dynamics and ParM assembly in Escherichia coli. Plasmids lacking a par operon undergo confined diffusion with a diffusion constant of 5 × 10−5 μm2/s and a confinement radius of 0.28 μm. Single par-containing plasmids also move diffusively but with a larger diffusion constant (4 × 10−4 μm2/s) and confinement radius (0.42 μm). ParM filaments are dynamically unstable in vivo and form spindles that link pairs of par-containing plasmids and drive them rapidly (3.1 μm/min) toward opposite poles of the cell. After reaching the poles, ParM filaments rapidly and completely depolymerize. After ParM disassembly, segregated plasmids resume diffusive motion, often encountering each other many times and undergoing multiple rounds of ParM-dependent segregation in a single cell cycle. We propose that in addition to driving segregation, the par operon enables plasmids to search space and find sister plasmids more effectively.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Type II pneumocyte hypertrophy without activation of surfactant biosynthesis after partial pneumonectomy

1993 ◽  
Vol 264 (2) ◽  
pp. L153-L159 ◽  
Author(s):  
B. D. Uhal ◽  
M. D. Etter
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Tritiated Thymidine ◽  
Cell Cycle Distribution ◽  
Type Ii ◽  
Adult Rats ◽  
Type Ii Pneumocyte ◽  
Significant Difference

Hypertrophic and normotrophic type II pneumocytes were isolated from pneumonectomized adult rats by unit gravity (1 g) sedimentation or by fluorescence-activated cell sorting (FACS). In vivo or in vitro, hypertrophic cells incorporated significantly more 5-bromo-2'-deoxyuridine or tritiated thymidine into acid-insoluble material than did normotrophic cells. By FACS analysis of cell subpopulations isolated by 1 g, > 97% of normotrophic cells had G0-phase DNA content. In contrast, the cell cycle distribution of hypertrophic cells was 75% G1, 5% S, and 20% G2/M phases. Rates of incorporation of tritiated choline into total cellular phosphatidylcholine (PC) were identical in type II cells isolated from normal or pneumonectomized rats. The intracellular contents of disaturated phosphatidylcholine (DSPC) and total PC, as well as the ratio of these two lipids, were the same in hypertrophic and normotrophic cells from pneumonectomized rats and in cells isolated from normal rats. No significant difference was observed in the rate at which hypertrophic or normotrophic cells incorporated choline into DSPC. These results demonstrate that type II pneumocyte hypertrophy after pneumonectomy reflects balanced cell growth secondary to cell cycle progression in vivo. The data also indicate that epithelial cell hypertrophy after pneumonectomy, in contrast to that which develops after more acute lung injury, occurs without activation of surfactant biosynthesis or storage.

scholarly journals Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane

2008 ◽  
Vol 191 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Martin Krehenbrink ◽  
Frédéric Pecorari ◽  
Anthony P. Pugsley
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Outer Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Sodium Dodecyl ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Ii Secretion System

ABSTRACT The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

scholarly journals A fuzzy-registration approach to track cell divisions in time-lapse fluorescence microscopy

2018 ◽  
Author(s):  
Saoirse Amarteifio ◽  
Todd Fallesen ◽  
Gunnar Pruessner ◽  
Giovanni Sena
Keyword(s):  
Fluorescence Microscopy ◽  
Moving Objects ◽  
Identity Management ◽  
Complex Dynamics ◽  
Time Lapse ◽  
High Temporal Resolution ◽  
Data Sets ◽  
Management Problem ◽  
3D Tracking

AbstractBackgroundParticle-tracking in 3D is an indispensable computational tool to extract critical information on dynamical processes from raw time-lapse imaging. This is particularly true with in vivo time-lapse fluorescence imaging in cell and developmental biology, where complex dynamics are observed at high temporal resolution. Common tracking algorithms used with time-lapse data in fluorescence microscopy typically assume a continuous signal where background, recognisable keypoints and independently moving objects of interest are permanently visible. Under these conditions, simple registration and identity management algorithms can track the objects of interest over time. In contrast, here we consider the case of transient signals and objects whose movements are constrained within a tissue, where standard algorithms fail to provide robust tracking.ResultsTo optimize 3D tracking in these conditions, we propose the merging of registration and tracking tasks into a fuzzy registration algorithm to solve the identity management problem. We describe the design and application of such an algorithm, illustrated in the domain of plant biology, and make it available as an open-source software implementation. The algorithm is tested on mitotic events in 4D data-sets obtained with light-sheet fluorescence microscopy on growing Arabidopsis thaliana roots expressing CYCB::GFP. We validate the method by comparing the algorithm performance against both surrogate data and manual tracking.ConclusionThis method fills a gap in existing tracking techniques, following mitotic events in challenging data-sets using transient fluorescent markers in unregistered images.

scholarly journals MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

2017 ◽  
Author(s):  
Peishan Yi ◽  
Chao Xie ◽  
Guangshuo Ou
Keyword(s):  
Cell Cycle ◽  
Sensory Neurons ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Genetic Screen ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Neuronal Cilia ◽  
Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

scholarly journals Functions of a GyrBA Fusion Protein and Its Interaction with QnrB and Quinolones

2015 ◽  
Vol 59 (11) ◽  
pp. 7124-7127 ◽  
Author(s):  
Chunhui Chen ◽  
Regis Villet ◽  
George A. Jacoby ◽  
David C. Hooper
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Dna Gyrase ◽  
Temperature Sensitive ◽  
Type Ii ◽  
Wild Type ◽  
Interacting Protein ◽  
Content Type ◽  
Negative Supercoiling

ABSTRACTIn order to study the interactions betweenEscherichia coliDNA gyrase and the gyrase interacting protein QnrBin vivo, we constructed agyrB-gyrAfusion and validated its ability to correct the temperature-sensitive growth ofgyrAandgyrBmutants. Like wild-typegyrA, thegyrB-gyrAfusion complemented a quinolone-resistantgyrAmutant to increase susceptibility. It functioned as an active type II topoisomerase, catalyzed negative supercoiling of DNA, was inhibited by quinolone, and was protected by QnrB.

scholarly journals Synergy of Topoisomerase and Structural-Maintenance-of-Chromosomes Proteins Creates a Universal Pathway to Simplify Genome Topology

2018 ◽  
Author(s):  
Enzo Orlandini ◽  
Davide Marenduzzo ◽  
Davide Michieletto
Keyword(s):  
Cell Cycle ◽  
Specific Substrate ◽  
Biological Processes ◽  
Type Ii ◽  
Synergistic Mechanism ◽  
A Cell ◽  
Structural Maintenance Of Chromosomes ◽  
Knots And Links ◽  
Smc Proteins

Topological entanglements severely interfere with important biological processes. For this reason, genomes must be kept unknotted and unlinked during most of a cell cycle. Type II Topoisomerase (TopoII) enzymes play an important role in this process but the precise mechanisms yielding systematic disentanglement of DNA in vivo are not clear. Here we report computational evidence that Structural Maintenance of Chromosomes (SMC) proteins – such as cohesins and condensins – can cooperate with TopoII to establish a synergistic mechanism to resolve topological entanglements. SMC-driven loop extrusion (or diffusion) induces the spatial localisation of essential crossings in turn catalysing the simplification of knots and links by TopoII enzymes even in crowded and confined conditions. The mechanism we uncover is universal in that it does not qualitatively depend on the specific substrate, whether DNA or chromatin, or on SMC processivity; we thus argue that this synergy may be at work across organisms and throughout the cell cycle.

Cortical actin movements during the first cell cycle of the Caenorhabditis elegans embryo

1996 ◽  
Vol 109 (2) ◽  
pp. 525-533 ◽  
Author(s):  
S. Hird
Keyword(s):  
Cell Cycle ◽  
Caenorhabditis Elegans ◽  
Fluorescence Microscopy ◽  
Actin Microfilaments ◽  
Cortical Actin ◽  
Daughter Cells ◽  
Actin Cortex ◽  
Asymmetrically Distributed ◽  
Cytoplasmic Determinants

The first division of the Caenorhabditis elegans embryo is unequal, generating daughter cells with distinct fates. The differences between the cells are believed to result from the partitioning of cytoplasmic determinants during the first cell cycle. Actin microfilaments play a critical, but poorly defined, role in this event. In this paper, the actin cortex in live embryos is studied during cytoplasmic localisation by fluorescently labelling microfilaments in oocytes and then using in vivo fluorescence microscopy to observe their behaviour. This reveals that there is a concerted movement of cortical actin to the anterior of the embryo at the time cytoplasmic localisation takes place. Furthermore, it is demonstrated that endogenous foci of F-actin are asymmetrically distributed following this event; these structures have previously been seen in fixed cortices. A model for the participation of the actin cytoskeleton in cytoplasmic localisation is presented based on these results.

Cell cycle in alveolar epithelial type II cells: integration of Matrigel and KGF

1997 ◽  
Vol 273 (3) ◽  
pp. L572-L580 ◽  
Author(s):  
S. Buckley ◽  
B. Driscoll ◽  
K. D. Anderson ◽  
D. Warburton
Keyword(s):  
Cell Cycle ◽  
Keratinocyte Growth Factor ◽  
Cell Cycle Control ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Type Ii Cells ◽  
Type Ii ◽  
Cell Cycle Regulators ◽  
Alveolar Epithelial

The regulation of cell cycle control in alveolar epithelial type II cells (AEC2) in response to peptide growth factors and extracellular matrix signals is not well understood. Herein, we have determined that, in adult rat AEC2 in primary culture on Engelbreth-Holm-Swarm biomatrix (Matrigel) in the presence of keratinocyte growth factor, the expression of key cell cycle control elements, including cyclins A and D and cyclin-dependent kinases (cdk) 1 and 4, is increased and that retinoblastoma protein (pRb) phosphorylation is also increased, with a corresponding decrease in the expression of p53 and the cdk inhibitors (cdkis) p21WAF1/CIP1 and p27KIP-1 compared with cells cultured on plastic. The Matrigel biomatrix-KGF culture conditions were also associated with an enhanced proliferative response, as measured by fluorescent-activated cell sorter analysis, thymidine incorporation into DNA, and proliferating cell nuclear antigen expression. This enhanced proliferation occurred with neither a soluble extract of Matrigel biomatrix nor with other simple biological matrices. We conclude that coordinated induction of key cyclins and cdks, with the concomitant suppression of key negative cell cycle regulators, occurs in AEC2 on Matrigel biomatrix in the presence of KGF. We speculate that the balance between cyclin and cdk activation and cdki suppression in AEC2 serves to integrate the combined influences of biomatrix and KGF signaling on pRb phosphorylation, thereby controlling transit through S phase of the cell cycle. Conversely, AEC2 express high levels of cdkis and p53 at rest in G1 phase. The latter finding may explain the quiescent state of normal adult AEC2 in vivo.

scholarly journals Lsr2, a nucleoid-associated protein influencing mycobacterial cell cycle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marta Kołodziej ◽  
Damian Trojanowski ◽  
Katarzyna Bury ◽  
Joanna Hołówka ◽  
Weronika Matysik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Biology ◽  
Fluorescent Microscopy ◽  
Regulation Of Gene Expression ◽  
Model Organism ◽  
Time Lapse ◽  
Mycobacterial Cell ◽  
Associated Proteins ◽  
Nucleoprotein Complexes

AbstractNucleoid-associated proteins (NAPs) are responsible for maintaining highly organized and yet dynamic chromosome structure in bacteria. The genus Mycobacterium possesses a unique set of NAPs, including Lsr2, which is a DNA-bridging protein. Importantly, Lsr2 is essential for the M. tuberculosis during infection exhibiting pleiotropic activities including regulation of gene expression (mainly as a repressor). Here, we report that deletion of lsr2 gene profoundly impacts the cell morphology of M. smegmatis, which is a model organism for studying the cell biology of M. tuberculosis and other mycobacterial pathogens. Cells lacking Lsr2 are shorter, wider, and more rigid than the wild-type cells. Using time-lapse fluorescent microscopy, we showed that fluorescently tagged Lsr2 forms large and dynamic nucleoprotein complexes, and that the N-terminal oligomerization domain of Lsr2 is indispensable for the formation of nucleoprotein complexes in vivo. Moreover, lsr2 deletion exerts a significant effect on the replication time and replisome dynamics. Thus, we propose that the Lsr2 nucleoprotein complexes may contribute to maintaining the proper organization of the newly synthesized DNA and therefore influencing mycobacterial cell cycle.

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