A century journey of organelles research in the plant endomembrane system.

We are entering an exciting century in the study of the plant organelles in the endomembrane system. Over the past century, especially within the past 50 years, tremendous advancements have been made in the complex plant cell to generate a much clearer and informative picture of plant organelles, including the molecular/morphological features, dynamic/spatial behavior, and physiological functions. Importantly, all these discoveries and achievements in the identification and characterization of organelles in the endomembrane system would not have been possible without: (1) the innovations and timely applications of various state-of-art cell biology tools and technologies for organelle biology research; (2) the continuous efforts in developing and characterizing new organelle markers by the plant biology community; and (3) the landmark studies on the identification and characterization of the elusive organelles. While molecular aspects and results for individual organelles have been extensively reviewed, the development of the techniques for organelle research in plant cell biology is less appreciated. As one of the ASPB Centennial Reviews on "organelle biology," here we aim to take a journey across a century of organelle biology research in plants by highlighting the important tools (or landmark technologies) and key scientists that contributed to visualize organelles. We then highlight the landmark studies leading to the identification and characterization of individual organelles in the plant endomembrane systems.

[Role of Liquid-Liquid Phase Separation in Cell Fate Transition and Diseases].

Liquid-liquid phase separation (LLPS), a novel mechanism of the organization and formation of cellular structures, plays a vital role in regulating cell fate transitions and disease pathogenesis and is gaining widespread attention. LLPS may lead to the assemblage of cellular structures with liquid-like fluidity, such as germ granules, stress granules, and nucleoli, which are classic membraneless organelles. These structures are typically formed through the high-concentration liquid aggregation of biomacromolecules driven by weak multivalent interactions. LLPS is involved in regulating various intracellular life activities and its dysregulation may cause the disruption of cellular functions, thereby contributing to the pathogenesis and development of neurodegenerative diseases, infectious diseases, cancers, etc. Herein, we summarized published findings on the LLPS dynamics of membraneless organelles in physiological and pathological cell fate transition, revealing their crucial roles in cell differentiation, development, and various pathogenic processes. This paper provides a fresh theoretical framework and potential therapeutic targets for LLPS-related studies, opening new avenues for future research.

Biomolecular condensates tunes immune signaling at the Host-Pathogen interface.

Membraneless organelles participate in diverse spatiotemporal regulation of cellular signal transduction by recruiting necessary signaling factors. During host-pathogen interactions, the plasma membrane (PM) at the interface between the plant and microbes serves as a central platform for forming multicomponent immune signaling hubs. The macromolecular condensation of the immune complex and regulators is important in regulating immune signaling outputs regarding strength, timing, and crosstalk between signaling pathways. This review discusses mechanisms that regulate specific and crosstalk of plant immune signal transduction pathways through macromolecular assembly and condensation.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Organelle Communication: Joined in Sickness and in Health.

Organelles are membrane-lined structures that compartmentalize subcellular biochemical functions. Therefore, interorganelle communication is crucial for cellular responses that require the coordination of such functions. Multiple principles govern interorganelle interactions, which arise from the complex nature of organelles: position, multilingualism, continuity, heterogeneity, proximity, and bidirectionality, among others. Given their importance, alterations in organelle communication have been linked to many diseases. Among the different types of contacts, endoplasmic reticulum mitochondria interactions are the best known; however, mounting evidence indicates that other organelles also have something to say in the pathophysiological conversation.

Robustness and Universality in Organelle Size Control.

One of the grand challenges in cellular biophysics is understanding the precision with which cells assemble and maintain subcellular structures. Organelle sizes, for example, must be flexible enough to allow cells to grow or shrink them as environments demand yet be maintained within homeostatic limits. Despite identification of molecular factors that regulate organelle sizes we lack insight into the quantitative principles underlying organelle size control. Here we show experimentally that cells can robustly control average fluctuations in organelle size. By demonstrating that organelle sizes obey a universal scaling relationship we predict theoretically, our framework suggests that organelles grow in random bursts from a limiting pool of building blocks. Burstlike growth provides a general biophysical mechanism by which cells can maintain on average reliable yet plastic organelle sizes.

Fine-tuning cell organelle dynamics during mitosis by small GTPases.

During mitosis, the allocation of genetic material concurs with organelle transformation and distribution. The coordination of genetic material inheritance with organelle dynamics directs accurate mitotic progression, cell fate determination, and organismal homeostasis. Small GTPases belonging to the Ras superfamily regulate various cell organelles during division. Being the key regulators of membrane dynamics, the dysregulation of small GTPases is widely associated with cell organelle disruption in neoplastic and non-neoplastic diseases, such as cancer and Alzheimer's disease. Recent discoveries shed light on the molecular properties of small GTPases as sophisticated modulators of a remarkably complex and perfect adaptors for rapid structure reformation. This review collects current knowledge on small GTPases in the regulation of cell organelles during mitosis and highlights the mediator role of small GTPase in transducing cell cycle signaling to organelle dynamics during mitosis.

Size regulation of multiple organelles competing for a limiting subunit pool.

How cells regulate the size of intracellular structures and organelles is a longstanding question. Recent experiments suggest that size control of intracellular structures is achieved through the depletion of a limiting subunit pool in the cytoplasm. While the limiting pool model ensures organelle-to-cell size scaling, it does not provide a mechanism for robust size control of multiple co-existing structures. Here we develop a generalized theory for size-dependent growth of intracellular structures to demonstrate that robust size control of multiple intracellular structures, competing for a limiting subunit pool, is achieved via a negative feedback between the growth rate and the size of the individual structure. This design principle captures size maintenance of a wide variety of subcellular structures, from cytoskeletal filaments to three-dimensional organelles. We identify the feedback motifs for structure size regulation based on known molecular processes, and compare our theory to existing models of size regulation in biological assemblies. Furthermore, we show that positive feedback between structure size and growth rate can lead to bistable size distribution and spontaneous size selection.

Hierarchical integration of mitochondrial and nuclear positioning pathways by the Num1 EF hand.

Positioning organelles at the right place and time is critical for their function and inheritance. In budding yeast, mitochondrial and nuclear positioning require the anchoring of mitochondria and dynein to the cell cortex by clusters of Num1. We have previously shown that mitochondria drive the assembly of cortical Num1 clusters, which then serve as anchoring sites for mitochondria and dynein. When mitochondrial inheritance is inhibited, mitochondrial-driven assembly of Num1 in buds is disrupted and defects in dynein-mediated spindle positioning are observed. Using a structure-function approach to dissect the mechanism of mitochondria-dependent dynein anchoring, we found that the EF hand-like motif (EFLM) of Num1 and its ability to bind calcium are required to bias dynein anchoring on mitochondria-associated Num1 clusters. Consistently, when the EFLM is disrupted, we no longer observe defects in dynein activity following inhibition of mitochondrial inheritance. Thus, the Num1 EFLM functions to bias dynein anchoring and activity in nuclear inheritance subsequent to mitochondrial inheritance. We hypothesize that this hierarchical integration of organelle positioning pathways by the Num1 EFLM contributes to the regulated order of organelle inheritance during the cell cycle.

Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

Imaging the living plant cell: From probes to quantification.

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

Fine-tuning cell organelle dynamics during mitosis by small GTPases / 医学前沿

During mitosis, the allocation of genetic material concurs with organelle transformation and distribution. The coordination of genetic material inheritance with organelle dynamics directs accurate mitotic progression, cell fate determination, and organismal homeostasis. Small GTPases belonging to the Ras superfamily regulate various cell organelles during division. Being the key regulators of membrane dynamics, the dysregulation of small GTPases is widely associated with cell organelle disruption in neoplastic and non-neoplastic diseases, such as cancer and Alzheimer's disease. Recent discoveries shed light on the molecular properties of small GTPases as sophisticated modulators of a remarkably complex and perfect adaptors for rapid structure reformation. This review collects current knowledge on small GTPases in the regulation of cell organelles during mitosis and highlights the mediator role of small GTPase in transducing cell cycle signaling to organelle dynamics during mitosis.

Intracellular thermometry uncovers spontaneous thermogenesis and associated thermal signaling.

Conventional thermal biology has elucidated the physiological function of temperature homeostasis through spontaneous thermogenesis and responses to variations in environmental temperature in organisms. In addition to research on individual physiological phenomena, the molecular mechanisms of fever and physiological events such as temperature-dependent sex determination have been intensively addressed. Thermosensitive biomacromolecules such as heat shock proteins (HSPs) and transient receptor potential (TRP) channels were systematically identified, and their sophisticated functions were clarified. Complementarily, recent progress in intracellular thermometry has opened new research fields in thermal biology. High-resolution intracellular temperature mapping has uncovered thermogenic organelles, and the thermogenic functions of brown adipocytes were ascertained by the combination of intracellular thermometry and classic molecular biology. In addition, intracellular thermometry has introduced a new concept, "thermal signaling", in which temperature variation within biological cells acts as a signal in a cascade of intriguing biological events.

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens.

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell's tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin's local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.

Transcription Regulators and Membraneless Organelles Challenges to Investigate Them.

Eukaryotic cells are composed of different bio-macromolecules that are divided into compartments called organelles providing optimal microenvironments for many cellular processes. A specific type of organelles is membraneless organelles. They are formed via a process called liquid-liquid phase separation that is driven by weak multivalent interactions between particular bio-macromolecules. In this review, we gather crucial information regarding different classes of transcription regulators with the propensity to undergo liquid-liquid phase separation and stress the role of intrinsically disordered regions in this phenomenon. We also discuss recently developed experimental systems for studying formation and properties of membraneless organelles.

Phase Separation and Mechanical Forces in Regulating Asymmetric Cell Division of Neural Stem Cells.

Asymmetric cell division (ACD) of neural stem cells and progenitors not only renews the stem cell population but also ensures the normal development of the nervous system, producing various types of neurons with different shapes and functions in the brain. One major mechanism to achieve ACD is the asymmetric localization and uneven segregation of intracellular proteins and organelles into sibling cells. Recent studies have demonstrated that liquid-liquid phase separation (LLPS) provides a potential mechanism for the formation of membrane-less biomolecular condensates that are asymmetrically distributed on limited membrane regions. Moreover, mechanical forces have emerged as pivotal regulators of asymmetric neural stem cell division by generating sibling cell size asymmetry. In this review, we will summarize recent discoveries of ACD mechanisms driven by LLPS and mechanical forces.

Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization.

Peptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones-critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.

Accurate model of liquid-liquid phase behavior of intrinsically disordered proteins from optimization of single-chain properties.

Many intrinsically disordered proteins (IDPs) may undergo liquid-liquid phase separation (LLPS) and participate in the formation of membraneless organelles in the cell, thereby contributing to the regulation and compartmentalization of intracellular biochemical reactions. The phase behavior of IDPs is sequence dependent, and its investigation through molecular simulations requires protein models that combine computational efficiency with an accurate description of intramolecular and intermolecular interactions. We developed a general coarse-grained model of IDPs, with residue-level detail, based on an extensive set of experimental data on single-chain properties. Ensemble-averaged experimental observables are predicted from molecular simulations, and a data-driven parameter-learning procedure is used to identify the residue-specific model parameters that minimize the discrepancy between predictions and experiments. The model accurately reproduces the experimentally observed conformational propensities of a set of IDPs. Through two-body as well as large-scale molecular simulations, we show that the optimization of the intramolecular interactions results in improved predictions of protein self-association and LLPS.

Microtubule-dependent subcellular organisation of pluripotent cells.

With the advancement of cutting-edge live imaging technologies, microtubule remodelling has evolved as an integral regulator for the establishment of distinct differentiated cells. However, despite their fundamental role in cell structure and function, microtubules have received less attention when unravelling the regulatory circuitry of pluripotency. Here, we summarise the role of microtubule organisation and microtubule-dependent events required for the formation of pluripotent cells in vivo by deciphering the process of early embryogenesis: from fertilisation to blastocyst. Furthermore, we highlight current advances in elucidating the significance of specific microtubule arrays in in vitro culture systems of pluripotent stem cells and how the microtubule cytoskeleton serves as a highway for the precise intracellular movement of organelles. This Review provides an informed understanding of the intrinsic role of subcellular architecture of pluripotent cells and accentuates their regenerative potential in combination with innovative light-inducible microtubule techniques.