Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Abstract

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Checkpoint Regulation of Nuclear Tos4 Defines S Phase Arrest in Fission Yeast

“…Overproduction leads to cell cycle delay (Vachon et al, 2013) . Of particularly interest to us, Tos4-GFP shows periodic accumulation in the nucleus coincident with S phase, consistent with its known regulation and the maturation timing of GFP (Kiang et al 2009;Bastos de Oliveira et al 2012;Escorcia et al 2019;Shen and Forsburg 2019). In fission yeast, this has been exploited in studies of cyclical re-replication induced by cyclin inhibition (Kiang et al 2010).…”

Checkpoint regulation of nuclear Tos4 defines S phase arrest in fission yeast

“…It is known to be regulated by the G1/S phase master transcription factor MBF (MluI-binding factor transcriptional complex) in both yeasts (Kiang et al 2009;). Tos4-GFP shows periodic accumulation in the nucleus coincident with S phase, consistent with its known regulation and the maturation timing of GFP (Kiang et al 2009;; Escorcia et al 2019;. In fission yeast, it has been used in studies of cyclical re-replication induced by cyclin inhibition (Kiang et al 2010).…”

A visual atlas of meiotic protein dynamics in living fission yeast

“…Rationale We used a uniform growth strategy to induce mating and meiosis between cells of opposite mating types, followed by imaging of cells on agar pads (see Methods (Escorcia and Forsburg, 2017; Escorcia et al, 2019)). Importantly, aside from the tagged genes, the strains are normal haploids that were induced to mate, followed directly by zygotic meiosis.…”

“…Additionally, many studies employ “snapshots” of different timepoints, rather than continuous observations with live cell imaging that reveal dynamic and brief events. We have established a standardized protocol for imaging meiosis over time in normal homothallic or heterothallic strains, which we employed previously (Escorcia et al, 2019). In this study, we chose representative proteins involved in different stages of meiosis, and compared their signal intensity, localization, and dynamics against a standard panel of meiotic nuclear events including karyogamy, horsetail formation, meiosis I and meiosis II divisions.…”

“…In particular, many studies employ a temperature sensitive pat1 mutant to drive a synchronous meiosis, even from haploids, but pat1 disrupts some aspects of meiosis (e.g., (Bähler et al, 1991; Iino and Yamamoto, 1985a; Yamamoto and Hiraoka, 2003). In this report, we examine protein dynamics in meiosis using consistent strains, growth conditions and a standardized imaging protocol (Escorcia et al, 2019; Green et al, 2015). This allows us to generate an atlas of meiotic protein behavior that will be a useful reference to many investigators studying this dynamic process.…”