Monitoring cell division rates in Medicago sativa roots by developmental live cell imaging

Live-cell imaging has revolutionized our understanding of dynamic cellular processes in bacteria and eukaryotes. While similar techniques have recently been applied to the study of halophilic archaea, our ability to explore the cell biology of thermophilic archaea is limited, due to the technical challenges of imaging at high temperatures. Here, we report the construction of the Sulfoscope, a heated chamber that enables live-cell imaging on an inverted fluorescent microscope. Using this system combined with thermostable fluorescent probes, we were able to image Sulfolobus cells as they divide, revealing a tight coupling between changes in DNA compaction, segregation and cytokinesis. By imaging deletion mutants, we observe important differences in the function of the two ESCRTIII proteins recently implicated in cytokinesis. The loss of CdvB1 compromises cell division, causing occasional division failures and fusion of the two daughter cells, whereas the deletion of cdvB2 leads to a profound loss of division symmetry, generating daughter cells that vary widely in size and eventually generating ghost cells. These data indicate that DNA separation and cytokinesis are coordinated in Sulfolobus, as is the case in eukaryotes, and that two contractile ESCRTIII polymers perform distinct roles to ensure that Sulfolobus cells undergo a robust and symmetrical division. Taken together, the Sulfoscope has shown to provide a controlled high temperature environment, in which cell biology of Sulfolobus can be studied in unprecedent details.

Download Full-text

Microtubule Probe for Correlative Super-Resolution Fluorescence and Electron Microscopy

10.1101/351098 ◽

2018 ◽

Cited By ~ 1

Author(s):

Xiaohe Tian ◽

Cesare De Pace ◽

Lorena Ruiz-Perez ◽

Bo Chen ◽

Rina Su ◽

...

Keyword(s):

Electron Microscopy ◽

Cell Division ◽

Live Cell Imaging ◽

Cell Imaging ◽

Super Resolution ◽

Synchronous Fluorescence ◽

Live Cell ◽

Fluorescence And Electron Microscopy ◽

20 Nm ◽

In Cells

We report a versatile cyclometalated Iridium (III) complex probe that achieves synchronous fluorescence-electron microscopy correlation to reveal microtubule ultrastructure in cells. The selective insertion of probe between repeated α and β units of microtubule triggers remarkable fluorescent enhancement, and high TEM contrast due to the presence of heavy Ir ions. The highly photostable probe allows live cell imaging of tubulin localization and motion during cell division with an resolution of 20 nm, and under TEM imaging reveals the αβ unit interspace of 45Å of microtubule in cells.

Download Full-text

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

10.1101/2021.09.15.460474 ◽

2021 ◽

Author(s):

Richard S Muniz ◽

Paul C Campbell ◽

Thomas E Sladewski ◽

Lars D Renner ◽

Christopher L de Graffenried

Keyword(s):

Cell Division ◽

Live Cell Imaging ◽

Cell Imaging ◽

Temporal Dynamics ◽

Live Cell ◽

Three Dimensions ◽

Imaging Method ◽

Differential Rate ◽

Pdms Stamp

Trypanosoma brucei, the causative agent of human African trypanosomiasis, employs a flagellum for dissemination within the parasite's mammalian and insect hosts. T. brucei cells are highly motile in culture and must be able to move in all three dimensions for reliable cell division. These characteristics have made long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of a variety of important cell-cycle events. To address this issue, we have devised an imaging approach that confines cells to small volumes that can be imaged continuously for up to 24 h. This system employs cast agarose microwells generated using a PDMS stamp that can be made with different dimensions to maximize cell viability and imaging quality. Using this approach, we have imaged individual T. brucei through multiple rounds of cell division with high spatial and temporal resolution. We have employed this method to study the differential rate of T. brucei daughter cell division and show that the approach is compatible with loss-of-function experiments such as small molecule inhibition and RNAi. We have also developed a strategy that employs in-well "sentinel" cells to monitor potential toxicity due to imaging. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.

Download Full-text

High-temperature live-cell imaging of cytokinesis, cell motility and cell-cell adhesion in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

10.1101/2020.02.16.951772 ◽

2020 ◽

Cited By ~ 2

Author(s):

Arthur Charles-Orszag ◽

Samuel J. Lord ◽

R. Dyche Mullins

Keyword(s):

Cell Division ◽

High Temperature ◽

Cell Motility ◽

Cell Biology ◽

Live Cell Imaging ◽

Cell Imaging ◽

Live Cell ◽

Sulfolobus Acidocaldarius ◽

Model Organisms ◽

Cell Cell

Significant technical challenges have limited the study of extremophile cell biology. For example, the absence of methods for performing high-resolution, live-cell imaging at high temperatures (>50°C) has impeded the study of cell motility and cell division in thermophilic archaea such as model organisms from the genus Sulfolobus. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.

Download Full-text

Plasmodium berghei kinesin-5 associates with the spindle apparatus during cell division and is important for efficient production of infectious sporozoites

10.1101/2020.07.03.186031 ◽

2020 ◽

Cited By ~ 2

Author(s):

Mohammad Zeeshan ◽

Declan Brady ◽

Rebecca R. Stanway ◽

Carolyn A. Moores ◽

Anthony A. Holder ◽

...

Keyword(s):

Cell Division ◽

Plasmodium Berghei ◽

Live Cell Imaging ◽

Cell Imaging ◽

Subcellular Location ◽

Live Cell ◽

Efficient Production ◽

Spindle Apparatus ◽

Partial Overlap ◽

Fixed Cell

AbstractKinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

Download Full-text

Cellulose synthase-like D (CSLD) proteins move in the plasma membrane and their targeting to cell tips, but not cell plates, depends on the actin cytoskeleton

10.1101/2021.08.19.457018 ◽

2021 ◽

Author(s):

Shu-Zon Wu ◽

Arielle M. Chaves ◽

Rongrong Li ◽

Magdalena Bezanilla ◽

Alison W. Roberts

Keyword(s):

Plasma Membrane ◽

Cell Division ◽

Live Cell Imaging ◽

Cell Imaging ◽

Tip Growth ◽

Cellulose Synthase ◽

Live Cell ◽

Cellulose Microfibrils ◽

Apical Plasma Membrane ◽

Cellulose Synthesis

Cellulose Synthase-Like D (CSLD) proteins are implicated in cell wall remodeling during tip growth and cell division in plants, and are known to generate β-1,4-glucan. It is unknown whether they form complexes and move in the plasma membrane like members of the Cellulose Synthase (CESA) family. We used the genetically tractable moss Physcomitrium patens, which has a filamentous protonemal stage that undergoes both tip growth and cell division and is amenable to high resolution live cell imaging, to investigate CSLD function and intracellular trafficking. CSLD2 and CSLD6 are highly expressed in gametophores and are redundantly required for gametophore cellular patterning. Live cell imaging revealed that CSLD6 is also expressed in protonemata where it moves in the plasma membrane and localizes to cell plates and cell tips. Notably, delivery to the apical plasma membrane, but not the cell plate, depends on actin. By comparing the behavior of endogenously tagged CSLD6 and CESA10, we discovered that CSLD6 movements in the plasma membrane were significantly faster, shorter in duration and less linear than CESA10 movements and were insensitive to the cellulose synthesis inhibitor isoxaben. These data suggest that CSLD6 and CESA10 function within different structures and may thus produce structurally distinct cellulose microfibrils.

Download Full-text