Microtubules in Candida albicans Hyphae Drive Nuclear Dynamics and Connect Cell Cycle Progression to Morphogenesis

ABSTRACT Candida albicans is an opportunistic fungal pathogen whose virulence is related to its ability to switch between yeast, pseudohyphal, and true-hyphal morphologies. To ask how long-distance nuclear migration occurs in C. albicans hyphae, we identified the fundamental properties of nuclear movements and microtubule dynamics using time-lapse microscopy. In hyphae, nuclei migrate to, and divide across, the presumptive site of septation, which forms 10 to 15 μm distal to the basal cell. The mother nucleus returns to the basal cell, while the daughter nucleus reiterates the process. We used time-lapse microscopy to identify the mechanisms by which C. albicans nuclei move over long distances and are coordinated with hyphal morphology. We followed nuclear migration and spindle dynamics, as well as the time and position of septum specification, defined it as the presumptum, and established a chronology of nuclear, spindle, and morphological events. Analysis of microtubule dynamics revealed that premitotic forward nuclear migration is due to the repetitive sliding of astral microtubules along the cell cortex but that postmitotic forward and reverse nuclear migrations are due primarily to spindle elongation. Free microtubules exhibit cell cycle regulation; they are present during interphase and disappear at the time of spindle assembly. Finally, a growth defect in strains expressing Tub2-green fluorescent protein revealed a connection between hyphal elongation and the nuclear cell cycle that is coordinated by hyphal length and/or volume.

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Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00808.x ◽

1998 ◽

Vol 28 (5) ◽

pp. 883-892 ◽

Cited By ~ 147

Author(s):

Chris D. Webb ◽

Peter L. Graumann ◽

Jason A. Kahana ◽

Aurelio A. Teleman ◽

Pamela A. Silver ◽

...

Keyword(s):

Cell Cycle ◽

Bacillus Subtilis ◽

Replication Origin ◽

Time Lapse ◽

Time Lapse Microscopy ◽

Rapid Movement ◽

Use Of Time ◽

Origin Region

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The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.

Blood ◽

10.1182/blood.v106.11.1653.1653 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1653-1653 ◽

Cited By ~ 1

Author(s):

Joseph E. Italiano ◽

Jennifer L. Richardson ◽

Harald Schulze ◽

Ksenija Drabek ◽

Chloe Bulinski ◽

...

Keyword(s):

Platelet Activation ◽

Immunofluorescence Microscopy ◽

Blood Platelet ◽

Time Lapse ◽

Microtubule Dynamics ◽

Cell Periphery ◽

Structural Studies ◽

Time Lapse Microscopy ◽

Marginal Band ◽

Tyrosinated Tubulin

Abstract The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.

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Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and cell death

10.1101/309914 ◽

2018 ◽

Author(s):

Zeynep Kabakci ◽

Simon Käppeli ◽

Giorgio Cozza ◽

Claudio Cantù ◽

Christiane König ◽

...

Keyword(s):

Cell Cycle ◽

Mixed Type ◽

Molecular Level ◽

Time Lapse ◽

Guided Discovery ◽

Enzymatic Assays ◽

Intestinal Crypt ◽

Time Lapse Microscopy ◽

Cycle Arrest ◽

Mechanism Of Inhibition

ABSTRACTCDC25 phosphatases have a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational approaches we defined a minimal common pharmacophore in established CDC25 inhibitors and performed a virtual screening of a proprietary library. Taking advantage of the availability of crystal structures for CDC25A and CDC25B and using a molecular docking strategy, we carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, studies on 3D organoids derived from intestinal crypt stem cells of Apc/K-Ras mice revealed that the compounds caused arrest of proliferation.

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Initiation of chromosome replication controls both division and replication cycles in E. coli through a double-adder mechanism

eLife ◽

10.7554/elife.48063 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 7

Author(s):

Guillaume Witz ◽

Erik van Nimwegen ◽

Thomas Julou

Keyword(s):

Cell Cycle ◽

Time Lapse ◽

Growth Conditions ◽

Replication Initiation ◽

Stochastic Simulations ◽

E Coli ◽

Time Lapse Microscopy ◽

Statistical Framework ◽

Wide Range ◽

Cell Cycle Models

Living cells proliferate by completing and coordinating two cycles, a division cycle controlling cell size and a DNA replication cycle controlling the number of chromosomal copies. It remains unclear how bacteria such as Escherichia coli tightly coordinate those two cycles across a wide range of growth conditions. Here, we used time-lapse microscopy in combination with microfluidics to measure growth, division and replication in single E. coli cells in both slow and fast growth conditions. To compare different phenomenological cell cycle models, we introduce a statistical framework assessing their ability to capture the correlation structure observed in the data. In combination with stochastic simulations, our data indicate that the cell cycle is driven from one initiation event to the next rather than from birth to division and is controlled by two adder mechanisms: the added volume since the last initiation event determines the timing of both the next division and replication initiation events.

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Optimization of Cell Cycle Measurement by Time-Lapse Microscopy

Methods in Cell Biology - Laboratory Methods in Cell Biology ◽

10.1016/b978-0-12-405914-6.00007-x ◽

2012 ◽

pp. 143-161 ◽

Cited By ~ 3

Author(s):

Gabor Nagy ◽

Gabor Kiraly ◽

Gaspar Banfalvi

Keyword(s):

Cell Cycle ◽

Time Lapse ◽

Time Lapse Microscopy

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Time‐Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single‐Cell Level

Current Protocols in Cytometry ◽

10.1002/0471142956.cy1204s64 ◽

2013 ◽

Vol 64 (1) ◽

Cited By ~ 2

Author(s):

Rachel J. Errington ◽

Sally C. Chappell ◽

Imtiaz A. Khan ◽

Nuria Marquez ◽

Marie Wiltshire ◽

...

Keyword(s):

Cell Cycle ◽

Single Cell ◽

Time Lapse ◽

Single Cell Level ◽

Cell Level ◽

Time Lapse Microscopy

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Astral Microtubule Dynamics in Yeast: A Microtubule-based Searching Mechanism for Spindle Orientation and Nuclear Migration into the Bud

The Journal of Cell Biology ◽

10.1083/jcb.139.4.985 ◽

1997 ◽

Vol 139 (4) ◽

pp. 985-994 ◽

Cited By ~ 206

Author(s):

Sidney L. Shaw ◽

Elaine Yeh ◽

Paul Maddox ◽

E.D. Salmon ◽

Kerry Bloom

Keyword(s):

Fluorescent Protein ◽

Dynamic Instability ◽

Dynamic Properties ◽

Nuclear Migration ◽

Cytoplasmic Dynein ◽

Microtubule Dynamics ◽

Spindle Orientation ◽

Cell Cortex ◽

Nuclear Movement ◽

Bud Neck

Localization of dynein–green fluorescent protein (GFP) to cytoplasmic microtubules allowed us to obtain one of the first views of the dynamic properties of astral microtubules in live budding yeast. Several novel aspects of microtubule function were revealed by time-lapse, three-dimensional fluorescence microscopy. Astral microtubules, about four to six in number for each pole, exhibited asynchronous dynamic instability throughout the cell cycle, growing at ≅0.3–1.5 μm/min toward the cell surface then switching to shortening at similar velocities back to the spindle pole body (SPB). During interphase, a conical array of microtubules trailed the SPB as the nucleus traversed the cytoplasm. Microtubule disassembly by nocodozole inhibited these movements, indicating that the nucleus was pushed around the interior of the cell via dynamic astral microtubules. These forays were evident in unbudded G1 cells, as well as in late telophase cells after spindle disassembly. Nuclear movement and orientation to the bud neck in S/G2 or G2/M was dependent on dynamic astral microtubules growing into the bud. The SPB and nucleus were then pulled toward the bud neck, and further microtubule growth from that SPB was mainly oriented toward the bud. After SPB separation and central spindle formation, a temporal delay in the acquisition of cytoplasmic dynein at one of the spindle poles was evident. Stable microtubule interactions with the cell cortex were rarely observed during anaphase, and did not appear to contribute significantly to spindle alignment or elongation into the bud. Alterations of microtubule dynamics, as observed in cells overexpressing dynein-GFP, resulted in eventual spindle misalignment. These studies provide the first mechanistic basis for understanding how spindle orientation and nuclear positioning are established and are indicative of a microtubule-based searching mechanism that requires dynamic microtubules for nuclear migration into the bud.

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The Gadd45 Family As Mediators of Hematopoietic Stem Cell Differentiation - from Genotoxic Stress to Cytokine-Induced Differentiation

Blood ◽

10.1182/blood.v126.23.1188.1188 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1188-1188

Author(s):

Susanne Wingert ◽

Frederic B Thalheimer ◽

Nadine Haetscher ◽

Maike Rehage ◽

Hubert Serve ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Stem Cell ◽

Stem Cell Differentiation ◽

Genotoxic Stress ◽

Time Lapse ◽

Cellular Systems ◽

Hematopoietic Stem ◽

Time Lapse Microscopy ◽

Differentiation Induction

Abstract The growth arrest and DNA-damage-inducible 45 (Gadd45) protein family is encoded by three genes, Gadd45a, b and g. All members of the family are early responders of cellular stress with tumor-suppressive function. In leukemia, the Gadd45 genes are often epigenetically silenced. Lately, we identified the GADD45 Gamma as the molecular link of differentiation-promoting cytokines to induce differentiation in HSCs (1). Here, we unraveled the function of the genotoxic stress-induced family member GADD45 Alpha (GADD45A) in hematopoiesis. GADD45A has been implicated in cell cycle control, cell death and senescence, as well as in DNA damage repair. In general, GADD45A provides cellular stability by either arresting the cell cycle progression until DNA damage is repaired or, in cases of fatal damage, by inducing apoptosis. However, the function of GADD45A in hematopoiesis remains highly controversial. We revealed the changes in murine HSC fate control orchestrated by the expression of GADD45A at single cell resolution using time-lapse microscopy-based HSC tracking. In contrast to other cellular systems, GADD45A expression neither caused a cell cycle arrest nor an alteration in the decision between cell survival and apoptosis in HSCs. Strikingly, GADD45A strongly induced and accelerated the differentiation program in HSCs. Continuous tracking of individual HSCs and their progeny via time-lapse microscopy elucidated that once GADD45A was expressed, HSCs differentiate into committed progenitors within 29 h. GADD45A-expressing HSCs failed to long-term reconstitute the blood of recipients by inducing multi-lineage differentiation in vivo. The differentiation induction by GADD45A was transmitted by activating p38 MAPK signaling, and allowed the generation of megakaryocytic-erythroid, myeloid and lymphoid lineages. These data indicate that genotoxic stress-induced GADD45A expression in HSCs prevents their fatal transformation by directing them into differentiation and thereby clearing them from the system. As the differentiation induction is conserved throughout the GADD45 family our study establishes this cell fate as an HSC-specific DNA-damage escape mechanism. Comparative analyses of the three proteins will further dissect the induced mechanisms at the molecular level. (1) Thalheimer, F.B., Wingert, S., De Giacomo, P., Haetscher, N., Rehage, M., Brill, B., Theis, F.J., Hennighausen, L., Schroeder, T., Rieger, M.A. Cytokine-Regulated GADD45G Induces Differentiation and Lineage Selection in Hematopoietic Stem Cells. Stem Cell Reports 3(1):34-43. (2014) Disclosures No relevant conflicts of interest to declare.

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