Biomolecular condensates in fungi are tuned to function at specific temperatures.

Temperature can impact every reaction and molecular interaction essential to a cell. For organisms that cannot regulate their own temperature, a major challenge is how to adapt to temperatures that fluctuate unpredictability and on variable timescales. Biomolecular condensation offers a possible mechanism for encoding temperature-responsiveness and robustness into cell biochemistry and organization. To explore this idea, we examined temperature adaptation in a filamentous-growing fungus called Ashbya gossypii that engages biomolecular condensates containing the RNA-binding protein Whi3 to regulate mitosis and morphogenesis. We collected wild isolates of Ashbya that originate in different climates and found that mitotic asynchrony and polarized growth, which are known to be controlled by the condensation of Whi3, are temperature sensitive. Sequence analysis in the wild strains revealed changes to specific domains within Whi3 known to be important in condensate formation. Using an in vitro condensate reconstitution assay we found that temperature impacts the relative abundance of protein to RNA within condensates and that this directly impacts the material properties of the droplets. Finally, we found that exchanging Whi3 genes between warm and cold isolates was sufficient to rescue some, but not all, condensate-related phenotypes. Together these data demonstrate that material properties of Whi3 condensates are temperature sensitive, that these properties are important for function, and that sequence optimizes properties for a given climate.

Measuring Cellular Ploidy In Situ by Light Microscopy.

Determining cellular DNA content is valuable in the study of numerous biological processes, including organ development and injury repair. While FACS analysis of dissociated cells is a widely used method for assaying ploidy in a tissue cell population, for many tissue samples, it is possible and convenient to measure ploidy in situ using light microscopy. Here, we present two protocols for measuring cellular ploidy in tissues. These protocols are based on our studies in Drosophila melanogaster, but these are applicable to other settings as well. We present example results from Drosophila hindgut, midgut, and wing imaginal disc as examples. The first protocol focuses on measuring DNA content from decondensed interphase nuclei, while the second protocol details the visualization of condensed chromosomes for ploidy determination, either from mitotic cells or from interphase cells with drug-induced chromosome condensation. These techniques can be completed in 1 day and require standard lab supplies as well as a fluorescence light microscope.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Cytoplasmic sharing through apical membrane remodeling.

Multiple nuclei sharing a common cytoplasm are found in diverse tissues, organisms, and diseases. Yet, multinucleation remains a poorly understood biological property. Cytoplasm sharing invariably involves plasma membrane breaches. In contrast, we discovered cytoplasm sharing without membrane breaching in highly resorptive Drosophila rectal papillae. During a six-hour developmental window, 100 individual papillar cells assemble a multinucleate cytoplasm, allowing passage of proteins of at least 62 kDa throughout papillar tissue. Papillar cytoplasm sharing does not employ canonical mechanisms such as incomplete cytokinesis or muscle fusion pore regulators. Instead, sharing requires gap junction proteins (normally associated with transport of molecules < 1 kDa), which are positioned by membrane remodeling GTPases. Our work reveals a new role for apical membrane remodeling in converting a multicellular epithelium into a giant multinucleate cytoplasm.

Most cells are self-contained ­ they have a cell membrane that delimits and therefore defines the cell, separating it from other cells and from its environment. But sometimes several cells interconnect and form collectives so they can pool their internal resources. Some of the best-known examples of this happen in animal muscle cells and in the placenta of mammals. These cell collectives share their cytoplasm ­ the fluid within the cell membrane that contains the cell organelles ­ in one of two ways. Cells can either remain linked instead of breaking away when they divide, or they can fuse their membranes with those of their neighbors. Working out how cells link to their neighbors is difficult when so few examples of cytoplasm sharing are available for study. One way to tackle this is to try and find undiscovered cell collectives in an animal that is already heavily studied in the lab, such as the fruit fly Drosophila melanogaster. Peterson et al. used a genetic system that randomly labels each cell of the developing fly with one of three fluorescent proteins. These proteins are big and should not move between cells unless they are sharing their cytoplasm. This means that any cell containing two or more different colors of fluorescent protein must be connected to at least one of its neighbors. The experiment revealed that the cells of the fruit fly rectum share their cytoplasm in a way never seen before. This sharing occurs at a consistent point in the development of the fruit fly and uses a different set of genes to those used by interconnecting cells in mammal muscles and placenta. These genes produce proteins that reshape the membranes of the cells and fit them with gap junctions ­ tiny pores that cross from one membrane to the next, allowing the passage of very small molecules. In this case, the gap junctions allowed the cells to share molecules much larger than seen before. The result is a giant cell membrane containing the cytoplasm and organelles of more than a hundred individual cells. These findings expand scientists' understanding of how cells in a tissue can share cytoplasm and resources. They also introduce a new tissue in the fruit fly that can be used in future studies of cytoplasm sharing. Relatives of fruit flies, including fruit pests and mosquitos, have similar cell structure to the fruit fly, which means that further investigations using this system could result in advances in agriculture or human health.

Unconventional Cell Division Cycles from Marine-Derived Yeasts.

Fungi have been found in every marine habitat that has been explored; however, the diversity and functions of fungi in the ocean are poorly understood. In this study, fungi were cultured from the marine environment in the vicinity of Woods Hole, MA, USA, including from plankton, sponge, and coral. Our sampling resulted in 35 unique species across 20 genera. We observed many isolates by time-lapse, differential interference contrast (DIC) microscopy and analyzed modes of growth and division. Several black yeasts displayed highly unconventional cell division cycles compared to those of traditional model yeast systems. Black yeasts have been found in habitats inhospitable to other life and are known for halotolerance, virulence, and stress resistance. We find that this group of yeasts also shows remarkable plasticity in terms of cell size control, modes of cell division, and cell polarity. Unexpected behaviors include division through a combination of fission and budding, production of multiple simultaneous buds, and cell division by sequential orthogonal septations. These marine-derived yeasts reveal alternative mechanisms for cell division cycles that seem likely to expand the repertoire of rules established from classic model system yeasts.

Interphase cohesin regulation ensures mitotic fidelity after genome reduplication.

To ensure faithful genome propagation, mitotic cells alternate one round of chromosome duplication with one round of chromosome separation. Chromosome separation failure thus causes genome reduplication, which alters mitotic chromosome structure. Such structural alterations are well documented to impair mitotic fidelity following aberrant genome reduplication, including in diseased states. In contrast, we recently showed that naturally occurring genome reduplication does not alter mitotic chromosome structure in Drosophila papillar cells. Our discovery raised the question of how a cell undergoing genome reduplication might regulate chromosome structure to prevent mitotic errors. Here, we show that papillar cells ensure mitotic fidelity through interphase cohesin regulation. We demonstrate a requirement for cohesins during programmed rounds of papillar genome reduplication known as endocycles. This interphase cohesin regulation relies on cohesin release but not cohesin cleavage and depends on the conserved cohesin regulator Pds5 . Our data suggest that a distinct form of interphase cohesin regulation ensures mitotic fidelity after genome reduplication.

Polyteny: still a giant player in chromosome research.

In this era of high-resolution mapping of chromosome territories, topological interactions, and chromatin states, it is increasingly appreciated that the positioning of chromosomes and their interactions within the nucleus is critical for cellular function. Due to their large size and distinctive structure, polytene chromosomes have contributed a wealth of knowledge regarding chromosome regulation. In this review, we discuss the diversity of polytene chromosomes in nature and in disease, examine the recurring structural features of polytene chromosomes in terms of what they reveal about chromosome biology, and discuss recent advances regarding how polytene chromosomes are assembled and disassembled. After over 130 years of study, these giant chromosomes are still powerful tools to understand chromosome biology.

Distinct responses to reduplicated chromosomes require distinct Mad2 responses.

Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention.

The French paradox: Determining the superoxide-scavenging capacity of red wine and other beverages.

Plant-derived phenolic compounds such as those found in red wine, tea, and certain fruit juices may protect against cardiovascular disease by detoxifying (scavenging) superoxide and other unstable reactive oxygen species. We present a laboratory exercise that can be used to assess the superoxide-scavenging capacity of beverages. Among the beverages examined, only those known to be rich in phenolic compounds (red wine, green tea, blueberry juice, and stout beer) exhibited appreciable superoxide scavenging. White wine and a 10% ethanol solution served as controls and did not scavenge superoxide.