A Universal and Programmable Platform based on Fluorescent Peptide-Conjugated Probes for Detection of Proteins in Organelles of Living Cells.

Realizing protein analysis in organelles of living cells is of great significance for developing diagnostic and therapeutic methods of diseases. Fluorescent-labeled antibodies with well imaging performance and high affinity are classical biochemical tools for protein analysis, while due to the inability to effectively enter into cells, not to mention organelles and the uncontrollable reaction sites that might cause antibodies inactivation when chemically modification, they are hard to apply to living cells. Inspired by the structure of fluorescent-labeled antibodies, we designed as a universal detection platform that was based on the peptide-conjugated probes (PCPs) and consisted of three parts: a) a rotor type fluorescent molecular scaffold for conjugation and signal output; b) the cell penetration protein recognition unit; c) the subcellular organelle targeting unit. In living cells, PCPs could firstly localize at organelles and then proceed protein specific recognition, thus jointly leading to the restriction of twisted intramolecular charge transfer and activation of fluorescence signal. As a proof-of-concept, six different proteins in three typical intracellular organelles could be detected by our platform through simply replacing the recognition sequence of proteins and matching organelle targeting units. The position and intensity of fluorescence signals demonstrated specificity of PCPs and universality of the platform.

MCSdb, a database of proteins residing in membrane contact sites.

Organelles do not act as autonomous discrete units but rather as interconnected hubs that engage in extensive communication by forming close contacts called "membrane contact sites (MCSs)". And many proteins have been identified as residing in MCS and playing important roles in maintaining and fulfilling specific functions within these microdomains. However, a comprehensive compilation of these MCS proteins is still lacking. Therefore, we developed MCSdb, a manually curated resource of MCS proteins and complexes from publications. MCSdb documents 7010 MCS protein entries and 263 complexes, involving 24 organelles and 44 MCSs across 11 species. Additionally, MCSdb orchestrates all data into different categories with multitudinous information for presenting MCS proteins. In summary, MCSdb provides a valuable resource for accelerating MCS functional interpretation and interorganelle communication deciphering.

Unraveling Ferroptosis Mechanisms: Tracking Cellular Viscosity with Small Molecular Fluorescent Probes.

Ferroptosis is a recently identified form of regulated cell death characterized by iron accumulation and lipid peroxidation. Numerous functions for ferroptosis have been identified in physiological as well as pathological processes, most notably in the treatment of cancer. The intricate balance of redox homeostasis is profoundly altered during ferroptosis, leading to alteration in cellular microenvironment. One such microenvironment is viscosity among others such as pH, polarity, and temperature. Therefore, understanding the dynamics of ferroptosis associated viscosity levels within organelles is crucial. To date, there are a very few reviews that detects ferroptosis assessing reactive species. In this review, we have summarized organelle's specific fluorescent probes that detects dynamics of microviscosity during ferroptosis. Also, we offer the readers an insight of their design strategy, photophysics and associated bioimaging concluding with the future perspective and challenges in the related field.

[Latest Findings on Phase Separation of Cytomechanical Proteins]. / ç»†èƒžåŠ›å­¦è›‹ç™½ç›¸åˆ†ç¦»çš„ç ”ç©¶è¿›å±•.

The cellular response to mechanical stimuli depends largely on the structure of the cell itself and the abundance of intracellular cytomechanical proteins also plays a key role in the response to the stimulation of external mechanical signals. Liquid-liquid phase separation (LLPS) is the process by which proteins or protein-RNA complexes spontaneously separate and form two distinct "phases", ie, a low-concentration phase coexisting with a high-concentration phase. According to published findings, membrane-free organelles form and maintain their structures and regulate their internal biochemical activities through LLPS. LLPS, a novel mechanism for intracellular regulation of the biochemical reactions of biomacromolecules, plays a crucial role in modulating the responses of cytomechanical proteins. LLPS leads to the formation of highly concentrated liquid-phase condensates through multivalent interactions between biomacromolecules, thereby regulating a series of intracellular life activities. It has been reported that a variety of cytomechanical proteins respond to external mechanical signals through LLPS, which in turn affects biological behaviors such as cell growth, proliferation, spreading, migration, and apoptosis. Herein, we introduced the mechanisms of cytomechanics and LLPS. In addition, we presented the latest findings on cytomechanical protein phase separation, covering such issues as the regulation of focal adhesion maturation and mechanical signal transduction by LIM domain-containing protein 1 (LIMD1) phase separation, the regulation of intercellular tight junctions by zonula occludens (ZO) phase separation, and the regulation of cell proliferation and apoptosis by cytomechanical protein phase separation of the Hippo signaling pathway. The proposition of LLPS provides an explanation for the formation mechanism of intracellular membraneless organelles and supplies new approaches to understanding the biological functions of intracellular physiology or pathology. However, the molecular mechanisms by which LLPS drives focal adhesions and cell-edge dynamics are still not fully understood. It is not clear whether LLPS under in vitro conditions can occur under physiological conditions of organisms. There are still difficulties to be overcome in using LLPS to explain the interactions of multiple intracellular molecules. Researchers should pursue answers to these questions in the future.

Dipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis.

Artificial organelles can manipulate cellular functions and introduce non-biological processes into cells. Coacervate droplets have emerged as a close analog of membraneless cellular organelles. Their biomimetic properties, such as molecular crowding and selective partitioning, make them promising components for designing cell-like materials. However, their use as artificial organelles has been limited by their complex molecular structure, limited control over internal microenvironment properties, and inherent colloidal instability. Here we report the design of dipeptide coacervates that exhibit enhanced stability, biocompatibility, and a hydrophobic microenvironment. The hydrophobic character facilitates the encapsulation of hydrophobic species, including transition metal-based catalysts, enhancing their efficiency in aqueous environments. Dipeptide coacervates carrying a metal-based catalyst are incorporated as active artificial organelles in cells and trigger an internal non-biological chemical reaction. The development of coacervates with a hydrophobic microenvironment opens an alternative avenue in the field of biomimetic materials with applications in catalysis and synthetic biology.

Liquid-Liquid phase separation in bacteria.

Cells are the essential building blocks of living organisms, responsible for carrying out various biochemical reactions and performing specific functions. In eukaryotic cells, numerous membrane organelles have evolved to facilitate these processes by providing specific spatial locations. In recent years, it has also been discovered that membraneless organelles play a crucial role in the subcellular organization of bacteria, which are single-celled prokaryotic microorganisms characterized by their simple structure and small size. These membraneless organelles in bacteria have been found to undergo Liquid-Liquid phase separation (LLPS), a molecular mechanism that allows for their assembly. Through extensive research, the occurrence of LLPS and its role in the spatial organization of bacteria have been better understood. Various biomacromolecules have been identified to exhibit LLPS properties in different bacterial species. LLPS which is introduced into synthetic biology applies to bacteria has important implications, and three recent research reports have shed light on its potential applications in this field. Overall, this review investigates the molecular mechanisms of LLPS occurrence and its significance in bacteria while also considering the future prospects of implementing LLPS in synthetic biology.

Mitochondria-targetable small molecule fluorescent probes for the detection of cancer-associated biomarkers: A review.

Cancer represents a global threat to human health, and effective strategies for improved cancer early diagnosis and treatment are urgently needed. The detection of tumor biomarkers has been one of the important auxiliary means for tumor screening and diagnosis. Mitochondria are crucial subcellular organelles that produce most chemical energy used by cells, control metabolic processes, and maintain cell function. Evidence suggests the close involvement of mitochondria with cancer development. As a consequence, the identification of cancer-associated biomarker expression levels in mitochondria holds significant importance in the diagnosis of early-stage diseases and the monitoring of therapy efficacy. Small-molecule fluorescent probes are effective for the identification and visualization of bioactive entities within biological systems, owing to their heightened sensitivity, expeditious non-invasive analysis and real-time detection capacities. The design principles and sensing mechanisms of mitochondrial targeted fluorescent probes are summarized in this review. Additionally, the biomedical applications of these probes for detecting cancer-associated biomarkers are highlighted. The limitations and challenges of fluorescent probes in vivo are also considered and some future perspectives are provided. This review is expected to provide valuable insights for the future development of novel fluorescent probes for clinical imaging, thereby contributing to the advancement of cancer diagnosis and treatment.

Self-Assembling Polypeptides in Complex Coacervation.

ConspectusIntracellular compartmentalization plays a pivotal role in cellular function, with membrane-bound organelles and membrane-less biomolecular "condensates" playing key roles. These condensates, formed through liquid-liquid phase separation (LLPS), enable selective compartmentalization without the barrier of a lipid bilayer, thereby facilitating rapid formation and dissolution in response to stimuli. Intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), which are often rich in charged and polar amino acid sequences, scaffold many condensates, often in conjunction with RNA.Comprehending the impact of IDP/IDR sequences on phase separation poses a challenge due to the extensive chemical diversity resulting from the myriad amino acids and post-translational modifications. To tackle this hurdle, one approach has been to investigate LLPS in simplified polypeptide systems, which offer a narrower scope within the chemical space for exploration. This strategy is supported by studies that have demonstrated how IDP function can largely be understood based on general chemical features, such as clusters or patterns of charged amino acids, rather than residue-level effects, and the ways in which these kinds of motifs give rise to an ensemble of conformations.Our laboratory has utilized complex coacervates assembled from oppositely charged polypeptides as a simplified material analogue to the complexity of liquid-liquid phase separated biological condensates. Complex coacervation is an associative LLPS that occurs due to the electrostatic complexation of oppositely charged macro-ions. This process is believed to be driven by the entropic gains resulting from the release of bound counterions and the reorganization of water upon complex formation. Apart from their direct applicability to IDPs, polypeptides also serve as excellent model polymers for investigating molecular interactions due to the wide range of available side-chain functionalities and the capacity to finely regulate their sequence, thus enabling precise control over interactions with guest molecules.Here, we discuss fundamental studies examining how charge patterning, hydrophobicity, chirality, and architecture affect the phase separation of polypeptide-based complex coacervates. These efforts have leveraged a combination of experimental and computational approaches that provide insight into molecular level interactions. We also examine how these parameters affect the ability of complex coacervates to incorporate globular proteins and viruses. These efforts couple directly with our fundamental studies into coacervate formation, as such "guest" molecules should not be considered as experiencing simple encapsulation and are instead active participants in the electrostatic assembly of coacervate materials. Interestingly, we observed trends in the incorporation of proteins and viruses into coacervates formed using different chain length polypeptides that are not well explained by simple electrostatic arguments and may be the result of more complex interactions between globular and polymeric species. Additionally, we describe experimental evidence supporting the potential for complex coacervates to improve the thermal stability of embedded biomolecules, such as viral vaccines.Ultimately, peptide-based coacervates have the potential to help unravel the physics behind biological condensates, while paving the way for innovative methods in compartmentalization, purification, and biomolecule stabilization. These advancements could have implications spanning medicine to biocatalysis.

[Progress and prospects of liquid-liquid phase separation in developmental diseases].

Membrane-bound organelles as well as membrane-free compartments exist in eukaryotic cells, which divide the nucleus and cytoplasm into distinct subregions and allow specific biochemical reactions to occur. The physiological mechanisms of membrane-bound organelles have been extensively characterized, but the formation and function of membrane-free compartments have not been thoroughly studied. Over the past decade, significant progress had been made in the studies about the role of liquid-liquid phase separation (LLPS) in the formation of membrane-free organelles. LLPS which serves as an aggregated separation mechanism for cellular biochemical reactions, is associated with a variety of physiological processes such as signal transduction and gene transcriptional regulation; while aberrant LLPS may contribute to the occurrence of developmental diseases. The present review investigates the role of LLPS as a mechanism of aggregation and segregation of cellular biochemical responses. The mechanisms of LLPS development and recent advances in the relationships between aberrant LLPS and developmental diseases are forward discussed, as well as how these advances may aid in the development of LLPS-based therapies.

Making the connection: How membrane contact sites have changed our view of organelle biology.

The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles in intracellular signaling, lipid metabolism, motor-protein-mediated membrane dynamics, organelle division, and organelle biogenesis. Here, we summarize the major conceptual changes that have occurred in cell biology as we have come to appreciate how contact sites integrate the activities of organelles.

Assembling membraneless organelles from de novo designed proteins.

Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological processes and augment natural systems. The bottom-up design of self-assembled objects such as microcompartments and membraneless organelles is one such challenge. Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli. To do this, we combine de novo α-helical sequences, intrinsically disordered linkers and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from arrested assemblies to dynamic condensates. The designs are characterized in cells and in vitro using biophysical methods and soft-matter physics. Finally, we use the designed polypeptide to co-compartmentalize a functional enzyme pair in E. coli, improving product formation close to the theoretical limit.

Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes.

SUMMARYAcidocalcisomes are organelles conserved during evolution and closely related to the so-called volutin granules of bacteria and archaea, to the acidocalcisome-like vacuoles of yeasts, and to the lysosome-related organelles of animal species. All these organelles have in common their acidity and high content of polyphosphate and calcium. They are characterized by a variety of functions from storage of phosphorus and calcium to roles in Ca2+ signaling, osmoregulation, blood coagulation, and inflammation. They interact with other organelles through membrane contact sites or by fusion, and have several enzymes, pumps, transporters, and channels.

Tools for assembling the cell: Towards the era of cell structural bioinformatics.

Cells consist of large components, such as organelles, that recursively factor into smaller systems, such as condensates and protein complexes, forming a dynamic multi-scale structure of the cell. Recent technological innovations have paved the way for systematic interrogation of subcellular structures, yielding unprecedented insights into their roles and interactions. In this workshop, we discuss progress, challenges, and collaboration to marshal various computational approaches toward assembling an integrated structural map of the human cell.

Bioorthogonal Chemistry in Cellular Organelles.

While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

Advances in mass spectrometry to unravel the structure and function of protein condensates.

Membrane-less organelles assemble through liquid-liquid phase separation (LLPS) of partially disordered proteins into highly specialized microenvironments. Currently, it is challenging to obtain a clear understanding of the relationship between the structure and function of phase-separated protein assemblies, owing to their size, dynamics and heterogeneity. In this Perspective, we discuss recent advances in mass spectrometry (MS) that offer several promising approaches for the study of protein LLPS. We survey MS tools that have provided valuable insights into other insoluble protein systems, such as amyloids, and describe how they can also be applied to study proteins that undergo LLPS. On the basis of these recent advances, we propose to integrate MS into the experimental workflow for LLPS studies. We identify specific challenges and future opportunities for the analysis of protein condensate structure and function by MS.

Sequence variations of phase-separating proteins and resources for studying biomolecular condensates.

Phase separation (PS) is an important mechanism underlying the formation of biomolecular condensates. Physiological condensates are associated with numerous biological processes, such as transcription, immunity, signaling, and synaptic transmission. Changes in particular amino acids or segments can disturb the protein's phase behavior and interactions with other biomolecules in condensates. It is thus presumed that variations in the phase-separating-prone domains can significantly impact the properties and functions of condensates. The dysfunction of condensates contributes to a number of pathological processes. Pharmacological perturbation of these condensates is proposed as a promising way to restore physiological states. In this review, we characterize the variations observed in PS proteins that lead to aberrant biomolecular compartmentalization. We also showcase recent advancements in bioinformatics of membraneless organelles (MLOs), focusing on available databases useful for screening PS proteins and describing endogenous condensates, guiding researchers to seek the underlying pathogenic mechanisms of biomolecular condensates.

Sunlight-directed fluorophore-switch in photosynthesis of cyanine subcellular organelle markers for bio-imaging.

The photoconvertible fluorophore synthesis enables the light controlled imaging channels switch for accurate tracking the quantity and localization of intracellular biomolecules in chemical biology. Herein, we repurposed the photochemistry of Fischer's base and developed a sunlight-directed fluorophore-switch strategy for high-efficiency trimethine cyanine (Cy3.5/Cy3) synthesis. The unexpected sunlight-directed photoconversion of Fischer's base proceeds in conventional solvents and accelerates in chloroform via photo-oxidation and hydrogen atom transfer without using extra additives, and the heterogenous dimerization mechanism was proposed and confirmed by isolation of the reactive intermediates. The reliable strategy is employed in the photosynthesis of commercially available cytomembrane marker (DiI) and other cyanine based organelle markers with appreciable yields. Sunlight-controlled fluorophore-switch of subcellular organelle markers in living cells validated the feasibility of our strategy with cell-tolerant character. Moreover, remote control synthesis of Cy3.5 in vivo directed via sunlight further demonstrated the extended application of our strategy. Therefore, this sunlight-directed strategy will facilitate exploitation of cyanine-based probes with switched fluorescence imaging channels and further enable precise description of the dynamic variations in living cells with minimal autofluorescence and cellular disturbance.

Theoretical and Data-Driven Approaches for Biomolecular Condensates.

Biomolecular condensation processes are increasingly recognized as a fundamental mechanism that living cells use to organize biomolecules in time and space. These processes can lead to the formation of membraneless organelles that enable cells to perform distinct biochemical processes in controlled local environments, thereby supplying them with an additional degree of spatial control relative to that achieved by membrane-bound organelles. This fundamental importance of biomolecular condensation has motivated a quest to discover and understand the molecular mechanisms and determinants that drive and control this process. Within this molecular viewpoint, computational methods can provide a unique angle to studying biomolecular condensation processes by contributing the resolution and scale that are challenging to reach with experimental techniques alone. In this Review, we focus on three types of dry-lab approaches: theoretical methods, physics-driven simulations and data-driven machine learning methods. We review recent progress in using these tools for probing biomolecular condensation across all three fields and outline the key advantages and limitations of each of the approaches. We further discuss some of the key outstanding challenges that we foresee the community addressing next in order to develop a more complete picture of the molecular driving forces behind biomolecular condensation processes and their biological roles in health and disease.

Protein phase separation: new insights into cell division.

As the foundation for the development of multicellular organisms and the self-renewal of single cells, cell division is a highly organized event which segregates cellular components into two daughter cells equally or unequally, thus producing daughters with identical or distinct fates. Liquid-liquid phase separation (LLPS), an emerging biophysical concept, provides a new perspective for us to understand the mechanisms of a wide range of cellular events, including the organization of membrane-less organelles. Recent studies have shown that several key organelles in the cell division process are assembled into membrane-free structures via LLPS of specific proteins. Here, we summarize the regulatory functions of protein phase separation in centrosome maturation, spindle assembly and polarity establishment during cell division.

Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles.

A variety of protein tags are available for genetically encoded protein labeling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and, finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example, within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.