Distinct Subcellular Structures Research Articles

Plasma membrane-associated ARAF condensates fuel RAS-related cancer drug resistance.

RAF protein kinases are major RAS effectors that function by phosphorylating MEK. Although all three RAF isoforms share a conserved RAS binding domain and bind to GTP-loaded RAS, only ARAF uniquely enhances RAS activity. Here we uncovered the molecular basis of ARAF in regulating RAS activation. The disordered N-terminal sequence of ARAF drives self-assembly, forming ARAF-RAS condensates tethered to the plasma membrane. These structures concentrate active RAS locally, impeding NF1-mediated negative regulation of RAS, thereby fostering receptor tyrosine kinase (RTK)-triggered RAS activation. In RAS-mutant tumors, loss of the ARAF N terminus sensitizes tumor cells to pan-RAF inhibition. In hormone-sensitive cancers, increased ARAF condensates drive endocrine therapy resistance, whereas ARAF depletion reverses RTK-dependent resistance. Our findings delineate ARAF-RAS protein condensates as distinct subcellular structures sustaining RAS activity and facilitating oncogenic RAS signaling. Targeting ARAF-RAS condensation may offer a strategy to overcome drug resistance in both wild-type and mutant ARAF-mediated scenarios.

Self-priming of Plk1 binding to BubR1 ensures accurate mitotic progression

Plk1 is a key mitotic kinase that localizes to distinct subcellular structures to promote accurate mitotic progression. Plk1 recruitment depends on direct interaction between polo-box domain (PBD) on Plk1 and PBD binding motif (PBD BM) on the interactors. However, recent study showed that PBD BM alone is not enough for stable binding between CENP-U and Plk1 highlighting the complexity of the interaction which warrants further investigation. An important interactor for Plk1 during mitosis is the checkpoint protein BubR1. Plk1 bound to BubR1 via PBD interaction with pT620 phosphorylates BubR1 S676/T680 to promote BubR1-PP2A/B56 interaction. The BubR1-PP2A/B56 complex counteracts the destablizing effect on kinetochore-microtubule attachments by mitotic kinases to promote mitotic progression. Here we show that Plk1 phosphorylates T600/T608 on BubR1 and the double phosphorylation is critical for BubR1-Plk1 interaction. A similar mechanism for Plk1-Bub1 interaction also exists indicating a general principle for Plk1 kinetochore recruitment through self-priming. Mechanistically preventing BubR1 T600/T608 phosphorylation impairs chromosome congression and checkpoint silencing by reducing Plk1 and PP2A/B56 binding to BubR1. Increasing the binding affinity towards Plk1 and PP2A/B56 in BubR1 through protein engineering bypasses the requirement of T600/T608 phosphorylation for mitotic progression. These results reveal a new layer of regulation for accurate mitotic progression.

Noncanonical Amino Acid Incorporation Modulates Condensate States of Intrinsically Disordered Proteins in Escherichia coli Cells.

Biomolecular condensates are distinct subcellular structures with on-demand material states and dynamics in living cells. However, strategies for modulating their material states and physicochemical properties are lacking. Here, we report a chemical strategy for modulating the condensate states of intrinsically disordered proteins in bacterial Escherichia coli cells. This is achieved by noncanonical amino acid (DOPA) incorporation into model resilin-like proteins (RLPs) to endow autonomous oxidative and coordinative cross-linking mechanisms. Biogenesis of spherical gel-like condensates is achieved upon heterologous expression of the DOPA-incorporated RLP in the cells at 30 °C. We reveal that liquid-liquid phase separation underlies the formation of liquid condensates, and this liquid-like state is metastable and its dynamics is compromised by the oxidative and coordinative cross-linkings that ultimately drive the liquid-to-gel transition. Therefore, this study has deepened our understanding of biomolecular condensation and offers a new chemical strategy to expand the landscape of condensation phenotypes of living cells.

Deep learning permits imaging of multiple structures with the same fluorophores

Deep learning permits imaging of multiple structures with the same fluorophores

Asymmetric response emerges between creation and disintegration of force-bearing subcellular structures as revealed by percolation analysis.

Cells dynamically remodel their internal structures by modulating the arrangement of actin filaments (AFs). In this process, individual AFs exhibit stochastic behavior without knowing the macroscopic higher-order structures they are meant to create or disintegrate, but the mechanism allowing for such stochastic process-driven remodeling of subcellular structures remains incompletely understood. Here we employ percolation theory to explore how AFs interacting only with neighboring ones without recognizing the overall configuration can nonetheless create a substantial structure referred to as stress fibers (SFs) at particular locations. We determined the interaction probabilities of AFs undergoing cellular tensional homeostasis, a fundamental property maintaining intracellular tension. We showed that the duration required for the creation of SFs is shortened by the increased amount of preexisting actin meshwork, while the disintegration occurs independently of the presence of actin meshwork, suggesting that the coexistence of tension-bearing and non-bearing elements allows cells to promptly transition to new states in accordance with transient environmental changes. The origin of this asymmetry between creation and disintegration, consistently observed in actual cells, is elucidated through a minimal model analysis by examining the intrinsic nature of mechano-signal transmission. Specifically, unlike the symmetric case involving biochemical communication, physical communication to sense environmental changes is facilitated via AFs under tension, while other free AFs dissociated from tension-bearing structures exhibit stochastic behavior. Thus, both the numerical and minimal models demonstrate the essence of intracellular percolation, in which macroscopic asymmetry observed at the cellular level emerges not from microscopic asymmetry in the interaction probabilities of individual molecules, but rather only as a consequence of the manner of the mechano-signal transmission. These results provide novel insights into the role of the mutual interplay between distinct subcellular structures with and without tension-bearing capability. Insight: Cells continuously remodel their internal elements or structural proteins in response to environmental changes. Despite the stochastic behavior of individual structural proteins, which lack awareness of the larger subcellular structures they are meant to create or disintegrate, this self-assembly process somehow occurs to enable adaptation to the environment. Here we demonstrated through percolation simulations and minimal model analyses that there is an asymmetry in the response between the creation and disintegration of subcellular structures, which can aid environmental adaptation. This asymmetry inherently arises from the nature of mechano-signal transmission through structural proteins, namely tension-mediated information exchange within cells, despite the stochastic behavior of individual proteins lacking asymmetric characters in themselves.

Divergent Physiological Functions of Four Atg22-like Proteins in Conidial Germination, Development, and Virulence of the Entomopathogenic Fungus Beauveria bassiana

In yeast, Atg22 functions as a vacuolar efflux transporter to release the nutrients from the vacuole to the cytosol after the degradation of autophagic bodies. There are more than one Atg22 domain-containing proteins in filamentous fungi, but their physiological roles are largely unknown. In this study, four Atg22-like proteins (BbAtg22A through D) were functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. These Atg22-like proteins exhibit different sub-cellular distributions. BbAtg22A localizes in lipid droplets. BbAtg22B and BbAtg22C are completely distributed in the vacuole, and BbAtg22D has an additional association with the cytomembrane. The ablation of Atg22-like proteins did not block autophagy. Four Atg22-like proteins systematically contribute to the fungal response to starvation and virulence in B. bassiana. With the exception of ∆Bbatg22C, the other three proteins contribute to dimorphic transmission. Additionally, BbAtg22A and BbAtg22D are required for cytomembrane integrity. Meanwhile, four Atg22-like proteins contribute to conidiation. Therefore, Atg22-like proteins link distinct sub-cellular structures for the development and virulence in B. bassiana. Our findings provide a novel insight into the non-autophagic roles of autophagy-related genes in filamentous fungi.

Super-multiplexing excitation spectral microscopy with multiple fluorescence bands.

Fluorescence microscopy, with high molecular specificity and selectivity, is a valuable tool for studying complex biological systems and processes. However, the ability to distinguish a large number of distinct subcellular structures in a single sample is impeded by the broad spectra of molecular fluorescence. We have recently shown that excitation spectral microscopy provides a powerful means to unmix up to six fluorophores in a single fluorescence band. Here, by working with multiple fluorescence bands, we extend this approach to the simultaneous imaging of up to ten targets, with the potential for further expansions. By covering the excitation/emission bandwidth across the full visible range, an ultra-broad 24-wavelength excitation scheme is established through frame-synchronized scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter (AOTF), so that full-frame excitation-spectral images are obtained every 24 camera frames, offering superior spectral information and multiplexing capability. With numerical simulations, we validate the concurrent imaging of 10 fluorophores spanning the visible range to achieve exceptionally low (∼0.5%) crosstalks. For cell imaging experiments, we demonstrate unambiguous identification of up to eight different intracellular structures labeled by common fluorophores of substantial spectral overlap with minimal color crosstalks. We thus showcase an easy-to-implement, cost-effective microscopy system for visualizing complex cellular components with more colors and lower crosstalks.

SUMO: Glue or Solvent for Phase-Separated Ribonucleoprotein Complexes and Molecular Condensates?

Spatial organization of cellular processes in membranous or membrane-less organelles (MLOs, alias molecular condensates) is a key concept for compartmentalizing biochemical pathways. Prime examples of MLOs are the nucleolus, PML nuclear bodies, nuclear splicing speckles or cytosolic stress granules. They all represent distinct sub-cellular structures typically enriched in intrinsically disordered proteins and/or RNA and are formed in a process driven by liquid-liquid phase separation. Several MLOs are critically involved in proteostasis and their formation, disassembly and composition are highly sensitive to proteotoxic insults. Changes in the dynamics of MLOs are a major driver of cell dysfunction and disease. There is growing evidence that post-translational modifications are critically involved in controlling the dynamics and composition of MLOs and recent evidence supports an important role of the ubiquitin-like SUMO system in regulating both the assembly and disassembly of these structures. Here we will review our current understanding of SUMO function in MLO dynamics under both normal and pathological conditions.

Chemical features of the photosensitizers new methylene blue N and S137 influence their subcellular localization and photoinactivation efficiency in Candida albicans

Chemical features of the photosensitizers new methylene blue N and S137 influence their subcellular localization and photoinactivation efficiency in Candida albicans

Uncovering the biology of myelin with optical imaging of the live brain.

Myelin has traditionally been considered a static structure that is produced and assembled during early developmental stages. While this characterization is accurate in some contexts, recent studies have revealed that oligodendrocyte generation and patterns of myelination are dynamic and potentially modifiable throughout life. Unique structural and biochemical properties of the myelin sheath provide opportunities for the development and implementation of multimodal label-free and fluorescence optical imaging approaches. When combined with genetically encoded fluorescent tags targeted to distinct cells and subcellular structures, these techniques offer a powerful methodological toolbox for uncovering mechanisms of myelin generation and plasticity in the live brain. Here, we discuss recent advances in these approaches that have allowed the discovery of several forms of myelin plasticity in developing and adult nervous systems. Using these techniques, long-standing questions related to myelin generation, remodeling, and degeneration can now be addressed.

Culture and Transfection of Zebrafish Primary Cells.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue. In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

Large‐scale high‐resolution micro‐XRF analysis of histological structures in the skin of the pigeon beak

Histological analysis of biological structures in the avian beak is performed by means of large‐scalehigh‐resolutionmicro‐X‐ray fluorescence analysis using a Maia detector system. Element maps of S, K, Ca, Fe and Zn of square millimeter sizes show distinct cellular and subcellular tissue structures in semi‐thin sections with a resolution at the (sub‐)micrometer scale. Non‐destructive qualitative and quantitative evaluation of the respective trace elements can be performed on the tissue samples. The elemental maps are compared with conventional differential histological stainings of parallel sections. This method is discussed as a pre‐examination tool for subsequent analysis of selected structural sites by diffractive and spectroscopic X‐ray nanoprobe techniques and, after additional ultrasectioning, also by transmission electron microscopy. Copyright © 2017 John Wiley & Sons, Ltd.

モータータンパク質による人工筋肉で駆動するマイクロロボットの開発

In living cells, motor proteins and cytoskeletons are ubiquitous, and involved in various cellular functions. The functions are performed through distinct self-organized subcellular structures, such as contractile rings and stress fibers. Inspired by this, we intended to develop a molecular system consisting of microtubules and kinesins which can self-organize into various morphologies. By using a genetically engineered kinesin, we found that microtubules and the kinesins were self-organized into contractile fibers. In this presentation, we will discuss the mechanism of formation of the contractile fiber, and their possible applications as a novel actuator for microdevices.

Clathrin localization and dynamics in Aspergillus nidulans.

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH-GFP) which localized to three distinct subcellular structures: late Golgi (trans-Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the "endocytic collar"); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin "hubs" dispersed in synchrony after the late Golgi marker PHOSBP . Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4-64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.

Loukoumasomes Are Distinct Subcellular Structures from Rods and Rings and Are Structurally Associated with MAP2 and the Nuclear Envelope in Retinal Cells.

“Rods and rings” (RR) and loukoumasomes are similarly shaped, subcellular macromolecular structures with as yet unknown function. RR, so named because of their shape, are formed in response to inhibition in the GTP or CTP synthetic pathways and are highly enriched in the two key enzymes of the nucleotide synthetic pathway. Loukoumasomes also occur as linear and toroidal bodies and were initially inferred to be the same as RR, largely due to their shared shape and size and the fact that it was unclear if they shared the same subcomponents. In human retinoblastoma tissue and cells we have observed toroidal, perinuclear, macromolecular structures of similar size and antigenicity to those previously reported in neurons (neuronal-loukoumasomes). To further characterize the subcomponents of the retinal-loukoumasomes, confocal analysis following immunocytochemical staining for alpha-tubulin, beta-III tubulin and detyrosinated tubulin was performed. These studies indicate that retinal-loukoumasomes are enriched for beta-III tubulin and other tubulins associated with microtubules. Immunofluorescence together with the in situ proximity ligation assay (PLA), confirmed that beta-III tubulin colocalized with detyrosinated tubulin within loukoumasomes. Our results indicate that these tissues contain only loukoumasomes because these macromolecular structures are immunoreactive with an anti-tubulin antibody but are not recognized by the prototype anti-RR/inosine monophosphate dehydrogenase (IMPDH) antibody (It2006). To further compare the RR and retinal-loukoumasomes, retinoblastoma cells were exposed to the IMPDH-inhibitor ribavirin, a drug known to induce the formation of RR. In contrast to RR, the production of retinal-loukoumasomes was unaffected. Coimmunostaining of Y79 cells for beta-III tubulin and IMPDH indicate that these cells, when treated with ribavirin, can contain both retinal-loukoumasomes and RR and that these structures are antigenically distinct. Subcellular fractionation studies indicate that ribavirin increased the RR subcomponent, IMPDH, in the nuclear fraction of Y79 cells from 21.3 ± 5.8% (0 mM ribavirin) to 122.8 ± 7.9% (1 mM ribavirin) while the subcellular localization of the retinal-loukoumasome subcomponent tubulin went unaltered. Further characterization of retinal-loukoumasomes in retinoblastoma cells reveals that they are intimately associated with lamin folds within the nuclear envelope. Using immunofluorescence and the in situ PLA in this cell type, we have observed colocalization of beta-III tubulin with MAP2. As MAP2 is a microtubule-associated protein implicated in microtubule crosslinking, this supports a role for microtubule crosslinkers in the formation of retinal-loukoumasomes. Together, these results suggest that loukoumasomes and RR are distinct subcellular macromolecular structures, formed by different cellular processes and that there are other loukoumasome-like structures within retinal tissues and cells.

Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies.

Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used. Antibodies to Sec6NT co-precipitated substantially more Sec5, -10, -15, Exo70 and -84 than did those to Sec6CT. In contrast, antibodies to Sec6CT co-precipitated more Sec3 and Sec8 than did those to Sec6NT. These results are consistent with a model in which exocyst activation during periods of rapid membrane expansion is accompanied by molecular rearrangements within the holocomplex or association with accessory proteins, which expose the Sec6 C-terminal domain when the complex is membrane-bound and conceal it when the complex is cytoplasmic.

The role of long non-coding RNAs in neurodevelopment, brain function and neurological disease.

Long non-coding RNAs (lncRNAs) are transcripts with low protein-coding potential that represent a large proportion of the transcriptional output of the cell. Many lncRNAs exhibit features indicative of functionality including tissue-restricted expression, localization to distinct subcellular structures, regulated expression and evolutionary conservation. Some lncRNAs have been shown to associate with chromatin-modifying activities and transcription factors, suggesting that a common mode of action may be to guide protein complexes to target genomic loci. However, the functions (if any) of the vast majority of lncRNA transcripts are currently unknown, and the subject of investigation. Here, we consider the putative role(s) of lncRNAs in neurodevelopment and brain function with an emphasis on the epigenetic regulation of gene expression. Associations of lncRNAs with neurodevelopmental/neuropsychiatric disorders, neurodegeneration and brain cancers are also discussed.

Ultrastructural Characterization and Three-Dimensional Architecture of Replication Sites in Dengue Virus-Infected Mosquito Cells

During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

Integrating cAMP and phospholipid signaling: role of phosphodiesterase 3B

Cyclic AMP (cAMP) is a ubiquitous intracellular messenger that acts to integrate and translate the information encoded by extracellular messenger molecules, including hormones and neurotransmitters. Indeed, numerous extracellular messengers act to modulate cellular functions by altering the intracellular level of cAMP through increasing the rate of cAMP synthesis by adenylyl cyclases (ACs) or the rate of cAMP hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). While early models proposed that cAMP was uniformly distributed throughout the cell, recent evidence is consistent with the idea that anchoring PDEs to selective subcellular locals may allow control of individual isolated subcellular pools of cAMP. In these studies, we demonstrate that human PDE3B can be targeted to distinct subcellular structures involved in regulating cAMP effects in hematopoietic cell lines, and that this signaling complex regulates the activity of a cAMP-activated phospholipase C, PLC-epsilon. In addition, this work is consistent with the novel idea that the regulation of PLC-epsilon by PDE3B is mediated through effects on the activity of the exchange proteins activated by cAMP (EPACs), and not the more prototyipcal cAMP effector Protein Kinse A (PKA). Our data are presented in the context of PDE3B expression within a multi-protein cellular complex and indicates PDE-inhibitor effects in these cells. Funded by HSFO & CIHR

Alternative splicing generates a novel non-secretable cytosolic isoform of NELL2

Alternative splicing generates a novel non-secretable cytosolic isoform of NELL2