Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

Circadian Organelles: Rhythms at All Scales

Circadian clocks have evolved in most light-sensitive organisms, from unicellular organisms to mammals. Consequently, a myriad of biological functions exhibits circadian rhythmicity, from behavior to physiology, through tissue and cellular functions to subcellular processes. Circadian rhythms in intracellular organelles are an emerging and exciting research arena. We summarize herein the current literature for rhythmicity in major intracellular organelles in mammals. These include changes in the morphology, content, and functions of different intracellular organelles. While these data highlight the presence of rhythmicity in these organelles, a gap remains in our knowledge regarding the underlying molecular mechanisms and their functional significance. Finally, we discuss the importance and challenges faced by spatio-temporal studies on these organelles and speculate on the presence of oscillators in organelles and their potential mode of communication. As circadian biology has been and continues to be studied throughout temporal and spatial axes, circadian organelles appear to be the next frontier.

Intertwined and Finely Balanced: Endoplasmic Reticulum Morphology, Dynamics, Function, and Diseases

The endoplasmic reticulum (ER) is an organelle that is responsible for many essential subcellular processes. Interconnected narrow tubules at the periphery and thicker sheet-like regions in the perinuclear region are linked to the nuclear envelope. It is becoming apparent that the complex morphology and dynamics of the ER are linked to its function. Mutations in the proteins involved in regulating ER structure and movement are implicated in many diseases including neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and amyotrophic lateral sclerosis (ALS). The ER is also hijacked by pathogens to promote their replication. Bacteria such as Legionella pneumophila and Chlamydia trachomatis, as well as the Zika virus, bind to ER morphology and dynamics-regulating proteins to exploit the functions of the ER to their advantage. This review covers our understanding of ER morphology, including the functional subdomains and membrane contact sites that the organelle forms. We also focus on ER dynamics and the current efforts to quantify ER motion and discuss the diseases related to ER morphology and dynamics.

License to Regulate: Noncoding RNA Special Agents in Plant Meiosis and Reproduction

The complexity of the subcellular processes that take place during meiosis requires a significant remodeling of cellular metabolism and dynamic changes in the organization of chromosomes and the cytoskeleton. Recently, investigations of meiotic transcriptomes have revealed additional noncoding RNA factors (ncRNAs) that directly or indirectly influence the course of meiosis. Plant meiosis is the point at which almost all known noncoding RNA-dependent regulatory pathways meet to influence diverse processes related to cell functioning and division. ncRNAs have been shown to prevent transposon reactivation, create germline-specific DNA methylation patterns, and affect the expression of meiosis-specific genes. They can also influence chromosome-level processes, including the stimulation of chromosome condensation, the definition of centromeric chromatin, and perhaps even the regulation of meiotic recombination. In many cases, our understanding of the mechanisms underlying these processes remains limited. In this review, we will examine how the different functions of each type of ncRNA have been adopted in plants, devoting attention to both well-studied examples and other possible functions about which we can only speculate for now. We will also briefly discuss the most important challenges in the investigation of ncRNAs in plant meiosis.

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha

Gametogenesis is an essential biological event for sexual reproduction in various organisms. Bryophytes and some other plants employ motile sperms (spermatozoids) as male gametes, which self-locomote to the egg cells to accomplish fertilization. Spermatozoids of bryophytes harbor distinctive morphological characteristics, including the cell body with a helical slender shape and two motile flagella at the anterior edge. During transformation from a spermatid to spermatozoid (spermiogenesis), the shape and cellular contents of spermatids are dynamically reorganized. However, how each organelle is reorganized during plant spermiogenesis remains obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to cellular and nuclear shapes and flagella development. We then examined the remodeling of microtubules and reorganization of endomembrane organelles during spermiogenesis. The results indicate that the state of post-translational modification of tubulin is dynamically changed during the formation of the flagella and spline, and the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner during spermiogenesis. These findings are expected to provide useful indexes to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Circulatory systems and mortality rates

Aging is a complex process involving multiple factors and subcellular processes, ultimately leading to the death of an organism. The microscopic processes that cause aging are relatively well understood and effective macroscopic theories help explain the universality of aging in complex systems. However, these theories fail to explain the diversity of aging observed for various lifeforms. As such, more complete "mesoscopic" theories of aging are needed, combining the biophysical details of microscopic failure and the macroscopic structure of complex systems. Here we explore two models: (1) a network theoretic model, and (2) a convection diffusion model emphasizing the biophysical details of communicated signals. The first model allows us to explore the effects of connectivity structures on aging. In our second model, cells interact through cooperative and antagonistic factors. We find by varying the ratio at which these factors affect cell death, as well as the reaction kinetics, diffusive and flow parameters, we obtain a wide diversity of mortality curves. As such, the connectivity structures as well as the biophysical details of how various factors are transported in an organism may explain the diversity of aging observed across different lifeforms.

Deep Learning-based Subcellular Phenotyping of Protrusion Dynamics Reveals Fine Differential Drug Responses at Subcellular and Single-Cell Levels

Intracellular processes such as cytoskeletal organization and organelle dynamics exhibit massive subcellular heterogeneity. Although recent advances in fluorescence microscopy allow researchers to acquire an unprecedented amount of live cell image data at high spatiotemporal resolutions, the traditional ensemble-averaging of uncharacterized subcellular heterogeneity could mask important activities. Moreover, the curse of dimensionality of these complex dynamic datasets prevents access to critical mechanistic details of subcellular processes. Here, we establish an unsupervised machine learning framework called DeepHACKS (Deep phenotyping of Heterogeneous Activities in the Coordination of cytosKeleton at the Subcellular level) for "deep phenotyping," which identifies rare subcellular phenotypes specifically sensitive to molecular perturbations. DeepHACKS dissects the heterogeneity of subcellular time-series datasets by allowing bi-directional LSTM (Long-Short Term Memory) neural networks to extract fine-grained temporal features by integrating autoencoders with conventional machine learning outcomes. We applied DeepHACKS to subcellular protrusion dynamics in pharmacologically perturbed epithelial cells, revealing fine differential responses of leading edge dynamics specific to each perturbation. Particularly, DeepHACKS in conjunction with blebistantin treatment revealed the emergence of rare subcellular and single-cell phenotypes driven by "bursting" protrusion. This suggests that the temporal features directly learned from leading edge dynamics enable fine-grained identification of drug-related phenotypes, which may not be accessible from static cell images. In summary, our study provides an analytical framework for detailed and quantitative understandings of molecular mechanisms hidden in their heterogeneity. DeepHACKS can be potentially applied to analyze various time-series data measured from other subcellular processes.

Mitochondria and the thermal limits of ectotherms

ABSTRACTTemperature is a critical abiotic factor shaping the distribution and abundance of species, but the mechanisms that underpin organismal thermal limits remain poorly understood. One possible mechanism underlying these limits is the failure of mitochondrial processes, as mitochondria play a crucial role in animals as the primary site of ATP production. Conventional measures of mitochondrial performance suggest that these organelles can function at temperatures much higher than those that limit whole-organism function, suggesting that they are unlikely to set organismal thermal limits. However, this conclusion is challenged by recent data connecting sequence variation in mitochondrial genes to whole-organism thermal tolerance. Here, we review the current state of knowledge of mitochondrial responses to thermal extremes and ask whether they are consistent with a role for mitochondrial function in shaping whole-organism thermal limits. The available data are fragmentary, but it is possible to draw some conclusions. There is little evidence that failure of maximal mitochondrial oxidative capacity as assessed in vitro sets thermal limits, but there is some evidence to suggest that temperature effects on ATP synthetic capacity may be important. Several studies suggest that loss of mitochondrial coupling is associated with the thermal limits for organismal growth, although this needs to be rigorously tested. Most studies have utilized isolated mitochondrial preparations to assess the effects of temperature on these organelles, and there remain many untapped opportunities to address these questions using preparations that retain more of their biological context to better connect these subcellular processes with whole-organism thermal limits.

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

The phenomenal epigenome in neurodevelopmental disorders

Disruption of chromatin structure due to epimutations is a leading genetic etiology of neurodevelopmental disorders, collectively known as chromatinopathies. We show that there is an increasing level of convergence from the high diversity of genes that are affected by mutations to the molecular networks and pathways involving the respective proteins, the disrupted cellular and subcellular processes, and their consequence for higher order cellular network function. This convergence is ultimately reflected by specific phenotypic features shared across the various chromatinopathies. Based on these observations, we propose that the commonly disrupted molecular and cellular anomalies might provide a rational target for the development of symptomatic interventions for defined groups of genetically distinct neurodevelopmental disorders.