Gerhold, Abigail et al. (2015) International Worm Meeting "Investigating the impact of organismal physiology and tissue architecture on mitosis by in situ live imaging of adult germline stem and progenitor cells."

Gerhold, Abigail, Larouche, Myreille, Labbe, Jean-Claude, Maddox, Paul

[International Worm Meeting, 2015]

The study of dynamic mitotic events, such as spindle assembly, chromosome segregation and cytokinesis, has been largely confined to tissue culture and embryonic model systems, which are readily amenable to live imaging. How these fundamental mitotic processes occur in adult stem and progenitor cells, which divide within the complex environment of a mature tissue and organism, remains largely unknown. We are using in situ live imaging of C. elegans germ line stem and progenitor cells to investigate various aspects of mitosis in cells dividing within their native environment in an intact animal. We have shown that, unlike in the early embryo, regulation of anaphase onset in germ cells by the spindle assembly checkpoint resembles that which has been described for classical tissue culture models. In addition the timing of anaphase entry is highly sensitive to dietary restriction, attenuated insulin signaling, germ line function and developmental stage. These results provide in vivo validation of models for spindle assembly checkpoint regulation developed in tissue culture systems and demonstrate that mitotic progression in an adult stem and progenitor cell population is highly sensitive to changes in organismal physiology. Germ cell mitosis also occurs within a complex tissue architecture - germ cells are partially syncytial and are arranged in a circumferential monolayer with syncytial openings facing a common cytoplasmic core. We are exploring how this tissue organization is maintained during cell division and how it influences ring closure during cytokinesis.

Cheng, Eric et al. (2021) International Worm Meeting "The role of the Insulin Signaling Pathway in C. elegans Germline Stem Cell Mitosis"

Gerhold, Abigail, Cheng, Eric

[International Worm Meeting, 2021]

The spindle assembly checkpoint (SAC) monitors kinetochore-microtubule attachments and halts mitotic progression in the presence of unattached or incorrectly attached chromosomes. The SAC ensures the fidelity of chromosome segregation during mitosis by guarding against chromosome mis-segregation and aneuploidy. While the events and mechanisms that control the SAC are relatively well understood, how they are influenced by developmental and environmental signaling networks remains unclear. The insulin signaling (IIS) pathway is a universally important and highly conserved pathway that integrates cell proliferation and development in many organisms. We are using C. elegans germline stem cells (GSCs) to ask how the IIS pathway affects mitotic duration and fidelity in vivo via the in situ live imaging of these mitotically dividing cells. This pathway has a known role in cell cycle progression, with daf-2 mutants having fewer germ cells and a lower mitotic index, compared to stage-matched N2 animals, due primarily to a delay in the G2 phase of the cell cycle. Removal of daf-18 or daf-16 restores normal germ cell numbers suggesting canonical signaling from DAF-2 to DAF-16 affecting the rate of cell cycle progression. While clear links have been established between IIS and GSC proliferation, IIS involvement in events during mitosis, including the SAC, remains unclear. Evidence from cell culture models suggests that IIS may play a role during mitosis; however, it is not clear whether this occurs under in vivo physiological conditions. We have found that daf-2 mutants have an increased duration of mitosis and decreased number of cells entering mitosis. Both effects can be rescued by subsequent deletion of daf-18 or daf-16. In addition, daf-2 mutants have an increased incidence of chromosome segregation errors when the SAC is compromised, suggesting impairment of spindle assembly. Ongoing work will assess whether these effects are cell autonomous and will determine the underlying cause of the observed increase in chromosome segregation errors.

Zellag Mohamed, Reda et al. (2019) International Worm Meeting "Physiological control of C. elegans germline stem cell mitosis."

Maddox, Paul, Gerhold, Abigail, Zellag Mohamed, Reda, Labbe, Jean-Claude

[International Worm Meeting, 2019]

Accurate segregation of the replicated genome is an essential step in the production of genetically identical daughter cells during mitosis. The fidelity of chromosome segregation relies upon robust spindle assembly and the activity of a surveillance system called the spindle assembly checkpoint. These mitotic processes have been studied extensively in tissue culture and embryonic model systems, which are readily amenable to live imaging, but which generally lack physiological complexity. As a result, how mitosis is adapted to accommodate in vivo physiological variability remains largely unknown. We are using in situ live imaging of C. elegans germ line stem cells (GSCs) to investigate various aspects of mitosis in cells dividing within the physiologically complex environment of an intact animal. We have shown that modulating dietary intake, Insulin signaling or reproductive activity influences mitotic progression in GSCs and may affect mitotic fidelity. In addition, we have found that GSCs have a stronger spindle assembly checkpoint than cells in the early embryo, a trait which may be linked to their germ cell identity. Here we will present an optimized method for in situ live imaging of GSCs and our results indicating that GSC mitosis is highly sensitive to changes in organismal physiology. We suggest that several features of mitotic progression are affected by the physiological context in which cell division occurs, which may help to explain why certain conditions predispose cells to the development of aneuploidy.

Zellag, Reda M. et al. (2021) International Worm Meeting "Deciphering the mechanism of mitotic spindle orientation in Caenorhabditis elegans germline stem cells"

Gerhold, Abigail R., Zhao, Yifan, Labbe, Jean-Claude, Poupart, Vincent, Singh, Ramya, Zellag, Reda M.

[International Worm Meeting, 2021]

Tissue-resident stem cells contribute to tissue development, homeostasis and repair in response to signals from a specialized microenvironment termed the niche. We are using Caenorhabditis elegans germline stem cells (GSCs), which are distinctly accessible for intravital imaging, as a model to elucidate how the interactions between stem cells, their niche and their tissue of residence regulate their division in situ. The orientation of cell division is controlled by the mitotic spindle and determines the position, and potentially the fate, of GSCs within the germline. However, the mechanisms that regulate mitotic spindle orientation in GSCs are not well known. Deciphering these mechanisms requires live-imaging of GSCs under physiological conditions. We have carried out a systematic investigation into the technical factors that impact GSC physiology during live imaging and have determined an optimized protocol for monitoring GSC divisions under minimally disruptive conditions. To permit large-scale analysis of spindle dynamics during GSC mitosis, we constructed CentTracker, an automated analysis tool, based on tracking and pairing of centrosomes, which allows a variety of mitotic parameters, including mitotic spindle orientation, to be rapidly assessed, and which is adaptable to other cell types in C. elegans and in other organisms. Analysis of a large dataset of dividing GSCs, indicated that GSCs have a strong spindle orientation bias towards the gonadal axis, that persists throughout mitosis, but do not exhibit spindle dynamics consistent with asymmetric cell division. We hypothesize that underlying cell or tissue anisotropies contribute to GSC spindle orientation and we are using CentTracker to identify and characterize these regulators. This work will contribute to our understanding of how GSCs are positioned within the germline during cell division, which may have implications for both GSC self-renewal and germline tissue organization.