Multiple Organelles Research Articles

A Review of Signaling Transduction Mechanisms in Osteoclastogenesis Regulation by Autophagy, Inflammation, and Immunity.

Osteoclastogenesis is an ongoing rigorous course that includes osteoclast precursors fusion and bone resorption executed by degradative enzymes. Osteoclastogenesis is controlled by endogenous signaling and/or regulators or affected by exogenous conditions and can also be controlled both internally and externally. More evidence indicates that autophagy, inflammation, and immunity are closely related to osteoclastogenesis and involve multiple intracellular organelles (e.g., lysosomes and autophagosomes) and certain inflammatory or immunological factors. Based on the literature on osteoclastogenesis induced by different regulatory aspects, emerging basic cross-studies have reported the emerging disquisitive orientation for osteoclast differentiation and function. In this review, we summarize the partial potential therapeutic targets for osteoclast differentiation and function, including the signaling pathways and various cellular processes.

Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.

A GGGGCC (G4 C2 ) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. Here, we performed live imaging of Drosophila motor neurons expressing expanded G4 C2 repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G4 C2 repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G4 C2 repeats. Thus, our data indicate that expanded G4 C2 repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.

Multiple organelle-targeted near-infrared fluorescent probes toward pH and viscosity

Organelles, including mitochondria (mito), lysosomes (lyso), endoplasmic reticulum (ER), Golgi apparatus (Golgi), and ribosome et al., play a vital role in maintaining the regular work of the cell. Viscosity is an essential parameter in the cellular microenvironment. Herein, four viscosity-sensitive near-infrared fluorescent probes DMPC, DEPC, DHDM and DHDV that can simultaneously target multiple organelles were synthesized. As the viscosity increased, the fluorescence intensity of the probes gradually increased due to the hindrance of the rotation of the carbon-carbon single bond. The fluorescence intensity of DHDV increased by about 453 times, and the fluorescence quantum yield also increased from 0.051 to 0.681. Cell experiments indicated the probes could simultaneously target four kinds of organelles, and the four probes could also track mitochondria with no dependence on membrane potential. Further experiments showed that the probes could detect viscosity changes in lyso and mito. In addition, the probes also demonstrated the advantages of low cytotoxicity, good anti-interference and stability, providing a simple and effective tool for studying the activity of organelles with changing viscosity signals.

NPC1 Deficiency Contributes to Autophagy-Dependent Ferritinophagy in HEI-OC1 Auditory Cells.

Niemann–Pick type C disease (NPCD) is a rare genetic syndrome characterized by cholesterol accumulation in multiple organelles. NPCD is mainly caused by gene deficiency of NPC intracellular cholesterol transporter 1 (NPC1). It has been reported that some of the NPCD patients exhibit clinical features of progressive hearing loss at high frequency and iron disorder, but the underlying relationship is unknown. A recent study has reported that ferroptosis contributes to the impairment of cochlear hair cells that are related to sensory hearing. In this study, we generated NPC1-deficient HEI-OC1 cells to show the effect of NPC1 deficiency on cochlear outer hair cells. We found that NPC1 deficiency enhances autophagy-dependent ferritinophagy to release Fe (II). Our work provides important insights into the effect of NPC1 deficiency in auditory cells, indicating that it induces ferroptosis and results in hearing loss.

Nestin-dependent mitochondria-ER contacts define stem Leydig cell differentiation to attenuate male reproductive ageing

Male reproductive system ageing is closely associated with deficiency in testosterone production due to loss of functional Leydig cells, which are differentiated from stem Leydig cells (SLCs). However, the relationship between SLC differentiation and ageing remains unknown. In addition, active lipid metabolism during SLC differentiation in the reproductive system requires transportation and processing of substrates among multiple organelles, e.g., mitochondria and endoplasmic reticulum (ER), highlighting the importance of interorganelle contact. Here, we show that SLC differentiation potential declines with disordered intracellular homeostasis during SLC senescence. Mechanistically, loss of the intermediate filament Nestin results in lower differentiation capacity by separating mitochondria-ER contacts (MERCs) during SLC senescence. Furthermore, pharmacological intervention by melatonin restores Nestin-dependent MERCs, reverses SLC differentiation capacity and alleviates male reproductive system ageing. These findings not only explain SLC senescence from a cytoskeleton-dependent MERCs regulation mechanism, but also suggest a promising therapy targeting SLC differentiation for age-related reproductive system diseases.

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection.

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

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.

Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay.

The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse® system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.

Promyelocytic leukemia nuclear body-like structures can assemble in mouse oocytes.

ABSTRACTPromyelocytic leukemia (PML) nuclear bodies (PML-NBs), a class of membrane-less cellular organelles, participate in various biological activities. PML-NBs are known as the core-shell-type nuclear body, harboring ‘client’ proteins in their core. Although multiple membrane-less organelles work in the oocyte nucleus, PML-NBs have been predicted to be absent from oocytes. Here, we show that some well-known PML clients (but not endogenous PML) co-localized with small ubiquitin-related modifier (SUMO) protein in the nucleolus and peri-centromeric heterochromatin of maturing oocytes. In oocytes devoid of PML-NBs, endogenous PML protein localized in the vicinity of chromatin. During and after meiotic resumption, PML co-localized with SUMO gathering around chromosomes. To examine the benefit of the PML-NB-free intranuclear milieu in oocytes, we deliberately assembled PML-NBs by microinjecting human PML-encoding plasmids into oocytes. Under conditions of limited SUMO availability, assembled PML-NBs tended to cluster. Upon proteotoxic stress, SUMO delocalized from peri-centromeric heterochromatin and co-localized with SC35 (a marker of nuclear speckles)-positive large compartments, which was disturbed by pre-assembled PML-NBs. These observations suggest that the PML-NB-free intranuclear environment helps reserve SUMO for emergent responses by redirecting the flux of SUMO otherwise needed to maintain PML-NB dynamics.

The endoplasmic reticulum membrane-bending protein RETICULON facilitates chloroplast relocation movement in Marchantia polymorpha.

Plant cells alter the intracellular positions of chloroplasts to ensure efficient photosynthesis, a process controlled by the blue light receptor phototropin. Chloroplasts migrate toward weak light (accumulation response) and move away from excess light (avoidance response). Chloroplasts are encircled by the endoplasmic reticulum (ER), which forms a complex network throughout the cytoplasm. To ensure rapid chloroplast relocation, the ER must alter its structure in conjunction with chloroplast relocation movement, but little is known about the underlying mechanism. Here, we searched for interactors of phototropin in the liverwort Marchantia polymorpha and identified a RETICULON (RTN) family protein; RTN proteins play central roles in ER tubule formation and ER network maintenance by stabilizing the curvature of ER membranes in eukaryotic cells. Marchantia polymorpha RTN1 (MpRTN1) is localized to ER tubules and the rims of ER sheets, which is consistent with the localization of RTNs in other plants and heterotrophs. The Mprtn1 mutant showed an increased ER tubule diameter, pointing to a role for MpRTN1 in ER membrane constriction. Furthermore, Mprtn1 showed a delayed chloroplast avoidance response but a normal chloroplast accumulation response. The live cell imaging of ER dynamics revealed that ER restructuring was impaired in Mprtn1 during the chloroplast avoidance response. These results suggest that during the chloroplast avoidance response, MpRTN1 restructures the ER network and facilitates chloroplast movement via an interaction with phototropin. Our findings provide evidence that plant cells respond to fluctuating environmental conditions by controlling the movements of multiple organelles in a synchronized manner.

Dicyanoisophorone derivatives with self-targeting abilities towards multiple organelles for fluorescent markers and viscosity detection

To improve self-targeting abilities towards organelles, four fluorescent dyes (1a-1d) were designed by connection of dicyanoisophorone to phenylpiperidinol (1a), naphthalenol (1b), indazole (1c) and pyrene (1d) unit with a double bond. Their absorption maxima were located at 423–517 nm; and emission maxima were at 565–682 nm with large Stokes shifts of 142–191 nm. Moreover, their fluorescence intensities increased 11–117 fold in glycerol compared to phosphate buffer solution, which might be due to the long excited state lifetimes (up to 1 ns) by transient absorption spectra. Furthermore, laser confocal experiments with HeLa cells proved that dyes 1a-1d were self-target fluorescent markers at 10–20 μM for lysosomes, endoplasmic reticulum, mitochondria and lipid droplets respectively; and they could be used for the fluorescent probes at 2 μM for viscosity detection within corresponding organelles by influence of ionophores. In addition, these dyes were able to detect viscosity changes in a Parkinson's disease model with PC-12 cells.

Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells

Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.

A Novel Peroxin is Required for Mitochondrial Morphology: Implications for Resolving a New Peroxisome‐Mitochondrial Contact Site

Peroxisomes are dynamic and ubiquitous organelles that house many metabolic pathways and interact with other organelles such as the endoplasmic reticulum, lipid droplets, and mitochondria. One mechanism for organelle interaction is through membrane contact sites. While contact sites between multiple organelles have been identified, little is known about the proteins that serve as molecular tethers in such sites. We study organelle dynamics using peroxisome‐like organelles called glycosomes in the early diverging organism Trypanosoma brucei and have identified a novel peroxin (protein involved in peroxisome biogenesis) that is essential for mitochondrial morphology. Silencing this protein leads to a significant growth defect and swollen mitochondria. Multiple mitochondrial membrane transport channels have been identified in immunoprecipitation studies. Based on these findings, we hypothesize that this protein that we have named a putative peroxisome‐mitochondrial contact protein (PPMCP), localizes to glycosomes and mitochondria at contact points, which facilitate the transfer of metabolites between the two organelles. Disruption of this connection results in “leaky” mitochondria and cell death. Current work is focused on resolving the metabolic defects in PPMCP‐deficient cells and identifying additional molecular components of these contact sites. This work forwards our understanding of how contact sites are established and the role they play in interorganelle communication.

PEX19 Coordinates Neutral Lipid Storage in Cells in a Peroxisome-Independent Fashion.

Cellular lipid metabolism is tightly regulated and requires a sophisticated interplay of multiple subcellular organelles to adapt to changing nutrient supply. PEX19 was originally described as an essential peroxisome biogenesis factor that selectively targets membrane proteins to peroxisomes. Metabolic aberrations that were associated with compromised PEX19 functions, were solely attributed to the absence of peroxisomes, which is also considered the underlying cause for Zellweger Spectrum Disorders. More recently, however, it was shown that PEX19 also mediates the targeting of the VCP/P97-recuitment factor UBXD8 to the ER from where it partitions to lipid droplets (LDs) but the physiological consequences remained elusive. Here, we addressed the intriguing possibility that PEX19 coordinates the functions of the major cellular sites of lipid metabolism. We exploited the farnesylation of PEX19 and deciphered the organelle-specific functions of PEX19 using systems level approaches. Non-farnesylated PEX19 is sufficient to fully restore the metabolic activity of peroxisomes, while farnesylated PEX19 controls lipid metabolism by a peroxisome-independent mechanism that can be attributed to sorting a specific protein subset to LDs. In the absence of this PEX19-dependent LD proteome, cells accumulate excess triacylglycerols and fail to fully deplete their neutral lipid stores under catabolic conditions, highlighting a hitherto unrecognized function of PEX19 in controlling neutral lipid storage and LD dynamics.

Proteomic characterization of post-mortem human brain tissue following ultracentrifugation-based subcellular fractionation.

Proteomic characterization of human brain tissue is increasingly utilized to identify potential novel biomarkers and drug targets for a variety of neurological diseases. In whole-tissue studies, results may be driven by changes in the proportion of the largest and most abundant organelles or tissue cell-type composition. Spatial proteomics approaches enhance our knowledge of disease mechanisms and changing signalling pathways at the subcellular level by taking into account the importance of cellular localization, which critically influences protein function. Density gradient-based ultracentrifugation methods allow for subcellular fractionation and have been utilized in cell lines, mouse and human brain tissue to quantify thousands of proteins in specific enriched organelles such as the pre- and post-synapse. Serial ultracentrifugation methods allow for the analysis of multiple cellular organelles from the same biological sample, and to our knowledge have not been previously applied to frozen post-mortem human brain tissue. The use of frozen human tissue for tissue fractionation faces two major challenges, the post-mortem interval, during which proteins may leach from their usual location into the cytosol, and freezing, which results in membrane breakdown. Despite these challenges, in this proof-of-concept study, we show that the majority of proteins segregate reproducibly into crude density-based centrifugation fractions, that the fractions are enriched for the appropriate organellar markers and that significant differences in protein localization can be observed between tissue from individuals with Alzheimer’s disease and control individuals.

Role of systems biology and multi-omics analyses in delineating spatial interconnectivity and temporal dynamicity of ER stress mediated cellular responses.

The endoplasmic reticulum (ER) is a membranous organelle involved in calcium storage, lipid biosynthesis, protein folding and processing. Many patho-physiological conditions and pharmacological agents are known to perturb normal ER function and can lead to ER stress, which severely compromise protein folding mechanism and hence poses high risk of proteotoxicity. Upon sensing ER stress, the different stress signaling pathways interconnect with each other and work together to preserve cellular homeostasis. ER stress response is a part of the integrative stress response (ISR) and might play an important role in the pathogenesis of chronic neurodegenerative diseases, where misfolded protein accumulation and cell death are common. The initiation, manifestation and progression of ER stress mediated unfolded protein response (UPR) is a complex procedure involving multiple proteins, pathways and cellular organelles. To understand the cause and consequences of such complex processes, implementation of an integrative holistic approach is required to identify novel players and regulators of ER stress. As multi-omics data-based systems analyses have shown potential to unravel the underneath molecular mechanism of complex biological systems, it is important to emphasize the utility of this approach in understanding the ER stress biology. In this review we first discuss the ER stress signaling pathways and regulatory players, along with their inter-connectivity. We next highlight the importance of systems and network biology approaches using multi-omics data in understanding ER stress mediated cellular responses. This report would help advance our current understanding of the multivariate spatial interconnectivity and temporal dynamicity of ER stress.

An Update on Coat Protein Complexes for Vesicle Formation in Plant Post-Golgi Trafficking.

Endomembrane trafficking is an evolutionarily conserved process for all eukaryotic organisms. It is a fundamental and essential process for the transportation of proteins, lipids, or cellular metabolites. The aforementioned cellular components are sorted across multiple membrane-bounded organelles. In plant cells, the endomembrane mainly consists of the nuclear envelope, endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network or early endosome (TGN/EE), prevacuolar compartments or multivesicular bodies (PVCs/MVBs), and vacuole. Among them, Golgi apparatus and TGN represent two central sorting intermediates for cargo secretion and recycling from other compartments by anterograde or retrograde trafficking. Several protein sorting machineries have been identified to function in these pathways for cargo recognition and vesicle assembly. Exciting progress has been made in recent years to provide novel insights into the sorting complexes and also the underlying sorting mechanisms in plants. Here, we will highlight the recent findings for the adaptor protein (AP) complexes, retromer, and retriever complexes, and also their functions in the related coated vesicle formation in post-Golgi trafficking.

Resolving variable cell viability-induced false negative: Accurate and high-contrast fluorescence diagnosis of cancer enabled by dual organelle targeting and multiple microenvironmental parameters responsive versatile carbon dots

Developing effective fluorescence-based diagnostic methods for cancer is of great importance but remains a major challenge owing to the low contrast and limited accuracy induced by the variable cell viability. To increase contrast and accuracy in cancer diagnosis, a dual-organelle-targeting and multiple-parameter-responsive design strategy is proposed here. As a proof of concept, versatile carbon dots (CDs) targeting mitochondria and nucleoli with ability to respond to three environmental parameters (mitochondrial membrane potential, viscosity, and RNA) are developed. These capabilities not only enable high-contrast and accurate imaging of cancer cells but also make the CDs highly robust against interference from environmental parameters alterations induced by cell damage or apoptosis. This new design strategy involving targeting of multiple organelles and multiple responsiveness to environmental parameters could inspire further research in bioactive material-based cancer imaging.

New insights into the pathogenesis of Hermansky–Pudlak syndrome

Hermansky-Pudlak syndrome (HPS) is characterized by defects of multiple tissue-specific lysosome-related organelles (LROs), typically manifesting with oculocutaneous albinism or ocular albinism, bleeding tendency, and in some cases with pulmonary fibrosis, inflammatory bowel disease or immunodeficiency, neuropsychological disorders. Eleven HPS subtypes in humans and at least 15subtypes in mice have been molecularly identified. Current understanding of the underlying mechanisms of HPS is focusing on the defective biogenesis of LROs. Compelling evidences have shown that HPS protein-associated complexes (HPACs) function in cargo transport, cargo recycling, and cargo removal to maintain LRO homeostasis. Further investigation on the molecular and cellular mechanism of LRO biogenesis and secretion will be helpful for better understanding of its pathogenesis and for the precise intervention of HPS.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.