Spindle architecture constrains karyotype in budding yeast.

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome number varies dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a set of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering, and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. However, with fewer than five centromeres, outward forces in the metaphase spindle cannot be countered by kinetochore-microtubule attachments, triggering mitotic defects. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

Experimental evolution for cell biology.

Evolutionary cell biology explores the origins, principles, and core functions of cellular features and regulatory networks through the lens of evolution. This emerging field relies heavily on comparative experiments and genomic analyses that focus exclusively on extant diversity and historical events, providing limited opportunities for experimental validation. In this opinion article, we explore the potential for experimental laboratory evolution to augment the evolutionary cell biology toolbox, drawing inspiration from recent studies that combine laboratory evolution with cell biological assays. Primarily focusing on approaches for single cells, we provide a generalizable template for adapting experimental evolution protocols to provide fresh insight into long-standing questions in cell biology.

Single-Cell RNA Sequencing in Yeast Using the 10× Genomics Chromium Device.

Single-cell RNA sequencing (scRNA-seq) is emerging as an essential technique for studying the physiology of individual cells in populations. Although well-established and optimized for mammalian cells, research of microorganisms has been faced with major technical challenges for using scRNA-seq, because of their rigid cell wall, smaller cell size and overall lower total RNA content per cell. Here, we describe an easy-to-implement adaptation of the protocol for the yeast Saccharomyces cerevisiae using the 10× Genomics platform, originally optimized for mammalian cells. Introducing Zymolyase, a cell wall-digesting enzyme, to one of the initial steps of single-cell droplet formation allows efficient in-droplet lysis of yeast cells, without affecting the droplet emulsion and further sample processing. In addition, we also describe the downstream data analysis, which combines established scRNA-seq analysis protocols with specific adaptations for yeast, and R-scripts for further secondary analysis of the data.

Gene Loss Predictably Drives Evolutionary Adaptation.

Loss of gene function is common throughout evolution, even though it often leads to reduced fitness. In this study, we systematically evaluated how an organism adapts after deleting genes that are important for growth under oxidative stress. By evolving, sequencing, and phenotyping over 200 yeast lineages, we found that gene loss can enhance an organism's capacity to evolve and adapt. Although gene loss often led to an immediate decrease in fitness, many mutants rapidly acquired suppressor mutations that restored fitness. Depending on the strain's genotype, some ultimately even attained higher fitness levels than similarly adapted wild-type cells. Further, cells with deletions in different modules of the genetic network followed distinct and predictable mutational trajectories. Finally, losing highly connected genes increased evolvability by facilitating the emergence of a more diverse array of phenotypes after adaptation. Together, our findings show that loss of specific parts of a genetic network can facilitate adaptation by opening alternative evolutionary paths.

Network hubs affect evolvability.

The regulatory processes in cells are typically organized into complex genetic networks. However, it is still unclear how this network structure modulates the evolution of cellular regulation. One would expect that mutations in central and highly connected modules of a network (so-called hubs) would often result in a breakdown and therefore be an evolutionary dead end. However, a new study by Koubkova-Yu and colleagues finds that in some circumstances, altering a hub can offer a quick evolutionary advantage. Specifically, changes in a hub can induce significant phenotypic changes that allow organisms to move away from a local fitness peak, whereas the fitness defects caused by the perturbed hub can be mitigated by mutations in its interaction partners. Together, the results demonstrate how network architecture shapes and facilitates evolutionary adaptation.