Rikkyo University

Sexual reproduction is a common mode of reproduction in many species. Gametes, which differentiate from germ cells, produce offspring after fertilization or conjugation. In animals and plants, eggs and sperms differentiate from germ cells, while in budding yeast, spores are formed within cells. In the latter process, a de novo membrane structure called prospore membrane is formed within the diploid cell, enveloping the nucleus produced during meiosis and resulting in the generation of four haploid spores. However, the mechanism underlying the formation of this new membrane structure has not yet been fully elucidated.

In the present study, live-cell imaging was employed to observe the process of meiosis and spore formation in detail, successfully capturing the moment when a membrane structure is formed inside a cell. The researchers focused on the secretory pathway which consists of the ER and the Golgi apparatus, transports proteins and lipids. They revealed that the number of ER exit sites, which serve as the starting point for membrane traffic, and the Golgi apparatus decrease during meiosis, but they reassemble during spore formation. The researchers’ search for molecules that control this process also led to the discovery of protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1. They found that in cells lacking Gip1, ER exit sites were not regenerated, resulting in prospore membranes of abnormal size. In other words, during spore formation cells have a mechanism for remodeling the secretory pathway, which enables the efficient transport of lipids for prospore membrane formation.

Some diseases related to human gamete formation and fertilization are caused by failures in membrane traffic. The results of the present study are expected to elucidate the mechanisms by which these diseases develop, potentially leading to new diagnostic and treatment approaches.

The cell is the basic unit of life in all organisms and is enclosed within a biological membrane (cell membrane) consisting of lipids and proteins. Inside cells, there are various organelles that are enclosed by biological membranes, each of which has its specific role and supports the cell's activities.

Many proteins produced in a cell are transported through intracellular membrane traffic^*1. ER and the Golgi apparatus are important organelles in this pathway. A wide variety of proteins and lipids produced in the ER, which are components of the cell membrane, are transported through specific regions of the ER membrane (ER exit sites) to the Golgi apparatus. The Golgi apparatus then sorts these proteins and lipids for delivery to their final destinations.

The life cycle of budding yeast, a eukaryote like humans (see Figure 1), includes the production of gametes through sexual reproduction. During meiosis and spore formation — gamete production in budding yeast — four new haploid cells, known as endospores, are produced inside a diploid cell (see Figure 2). In this process, new membrane structures are formed inside the diploid cell, enveloping the nucleus to create new cells, which are the spores. These spores will subsequently mature. However, it was unclear how membrane traffic, involving the ER and the Golgi apparatus, is controlled when supplying lipids to the new cell membranes at this stage.

The research team employed a method called live-cell imaging to conduct detailed observations of the movements of the cell membrane (prospore membrane), the ER, and the Golgi apparatus during spore formation. They achieved this by labeling each target with a different fluorescent protein. The results showed that the number of ER exit sites per cell decreased as meiosis progressed, but increased following the second meiotic division. The team then utilized super-resolution confocal live imaging microscopy (SCLIM)^*3 to examine the precise location of the ER exit sites within the cell and to observe their proliferation. During this process, the team simultaneously and three-dimensionally captured the movement of both the prospore membrane and the ER exit sites over time (see Figure 3). The results showed that the ER exit sites whose number in a cell decreased during meiosis are regenerated inside the newly formed prospore membranes.

The results indicated that the organelles involved in membrane traffic were significantly remodeled to specialize in the formation of new biological membranes (Figure 4). Moreover, this study suggests the presence of a signal transduction mechanism that regulates changes in this membrane traffic system.

The present study provides insights into the mechanisms of cell membrane formation during meiosis and spore formation, which are gamete formation in budding yeast. The molecular groups associated with the formation of ER exit sites, including Sec16, are conserved across species. In humans, defects in the intracellular transport system causes various diseases. In addition, any failure in gamete formation can lead to abnormalities in gamete development and infertility. The results of this study are expected to enhance our understanding of the mechanisms underlying diseases related to gamete formation and fertilization, as well as facilitating their diagnosis and treatment.

The present study was conducted as part of a research project funded by Grants-in-Aid for Scientific Research (21K06145, 23K05006, 18H05275, 22K06074) from the Japan Society for the Promotion of Science.