RESUMEN
The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.
Asunto(s)
Retículo Endoplásmico , Transducción de Señal , Retículo Endoplásmico/metabolismo , Neuronas/metabolismo , Calcio/metabolismo , Señalización del CalcioRESUMEN
The endoplasmic reticulum (ER) is a dynamic network of interconnected sheets and tubules that orchestrates the distribution of lipids, ions, and proteins throughout the cell. The impact of its complex, dynamic morphology on its function as an intracellular transport hub remains poorly understood. To elucidate the functional consequences of ER network structure and dynamics, we quantify how the heterogeneity of the peripheral ER in COS7 cells affects diffusive protein transport. In vivo imaging of photoactivated ER membrane proteins demonstrates their nonuniform spreading to adjacent regions, in a manner consistent with simulations of diffusing particles on extracted network structures. Using a minimal network model to represent tubule rearrangements, we demonstrate that ER network dynamics are sufficiently slow to have little effect on diffusive protein transport. Furthermore, stochastic simulations reveal a novel consequence of ER network heterogeneity: the existence of "hot spots" where sparse diffusive reactants are more likely to find one another. ER exit sites, specialized domains regulating cargo export from the ER, are shown to be disproportionately located in highly accessible regions, further from the outer boundary of the cell. Combining in vivo experiments with analytic calculations, quantitative image analysis, and computational modeling, we demonstrate how structure guides diffusive protein transport and reactions in the ER.
Asunto(s)
Retículo Endoplásmico , Proteínas , Cinética , Retículo Endoplásmico/metabolismo , Transporte de Proteínas , Transporte Biológico , Proteínas/metabolismoRESUMEN
Secretory cargo is recognized, concentrated and trafficked from endoplasmic reticulum (ER) exit sites (ERES) to the Golgi. Cargo export from the ER begins when a series of highly conserved COPII coat proteins accumulate at the ER and regulate the formation of cargo-loaded COPII vesicles. In animal cells, capturing live de novo cargo trafficking past this point is challenging; it has been difficult to discriminate whether cargo is trafficked to the Golgi in a COPII-coated vesicle. Here, we describe a recently developed live-cell cargo export system that can be synchronously released from ERES to illustrate de novo trafficking in animal cells. We found that components of the COPII coat remain associated with the ERES while cargo is extruded into COPII-uncoated, non-ER associated, Rab1 (herein referring to Rab1a or Rab1b)-dependent carriers. Our data suggest that, in animal cells, COPII coat components remain stably associated with the ER at exit sites to generate a specialized compartment, but once cargo is sorted and organized, Rab1 labels these export carriers and facilitates efficient forward trafficking.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Retículo Endoplásmico , Aparato de Golgi , Animales , Transporte Biológico , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Transporte de ProteínasRESUMEN
Mitochondria cannot be generated de novo; they must grow, replicate their genome, and divide in order to be inherited by each daughter cell during mitosis. Mitochondrial division is a structural challenge that requires the substantial remodelling of membrane morphology. Although division factors differ across organisms, the need for multiple constriction steps and a dynamin-related protein (Drp1, Dnm1 in yeast) has been conserved. In mammalian cells, mitochondrial division has been shown to proceed with at least two sequential constriction steps: the endoplasmic reticulum and actin must first collaborate to generate constrictions suitable for Drp1 assembly on the mitochondrial outer membrane; Drp1 then further constricts membranes until mitochondrial fission occurs. In vitro experiments, however, indicate that Drp1 does not have the dynamic range to complete membrane fission. In contrast to Drp1, the neuron-specific classical dynamin dynamin-1 (Dyn1) has been shown to assemble on narrower lipid profiles and facilitate spontaneous membrane fission upon GTP hydrolysis. Here we report that the ubiquitously expressed classical dynamin-2 (Dyn2) is a fundamental component of the mitochondrial division machinery. A combination of live-cell and electron microscopy in three different mammalian cell lines reveals that Dyn2 works in concert with Drp1 to orchestrate sequential constriction events that build up to division. Our work underscores the biophysical limitations of Drp1 and positions Dyn2, which has intrinsic membrane fission properties, at the final step of mitochondrial division.
Asunto(s)
Dinaminas/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Actinas/metabolismo , Animales , Línea Celular , Guanosina Trifosfato/metabolismo , Humanos , Hidrólisis , Mamíferos , Membranas Mitocondriales/metabolismoRESUMEN
Several organelles in eukaryotic cells, including mitochondria and the endoplasmic reticulum, form interconnected tubule networks extending throughout the cell. These tubular networks host many biochemical pathways that rely on proteins diffusively searching through the network to encounter binding partners or localized target regions. Predicting the behavior of such pathways requires a quantitative understanding of how confinement to a reticulated structure modulates reaction kinetics. In this work, we develop both exact analytical methods to compute mean first passage times and efficient kinetic Monte Carlo algorithms to simulate trajectories of particles diffusing in a tubular network. Our approach leverages exact propagator functions for the distribution of transition times between network nodes and allows large simulation time steps determined by the network structure. The methodology is applied to both synthetic planar networks and organelle network structures, demonstrating key general features such as the heterogeneity of search times in different network regions and the functional advantage of broadly distributing target sites throughout the network. The proposed algorithms pave the way for future exploration of the interrelationship between tubular network structure and biomolecular reaction kinetics.
RESUMEN
Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.
Asunto(s)
Calcio , Retículo Endoplásmico , Neuronas , Proteínas Nogo , Retículo Endoplásmico/metabolismo , Proteínas Nogo/metabolismo , Humanos , Calcio/metabolismo , Neuronas/metabolismo , Neuritas/metabolismo , Transporte Biológico , Proyección Neuronal/efectos de los fármacosRESUMEN
We investigate diffusive search on planar networks, motivated by tubular organelle networks in cell biology that contain molecules searching for reaction partners and binding sites. Exact calculation of the diffusive mean first-passage time on a spatial network is used to characterize the typical search time as a function of network connectivity. We find that global structural properties - the total edge length and number of loops - are sufficient to largely determine network exploration times for a variety of both synthetic planar networks and organelle morphologies extracted from living cells. For synthetic networks on a lattice, we predict the search time dependence on these global structural parameters by connecting with percolation theory, providing a bridge from irregular real-world networks to a simpler physical model. The dependence of search time on global network structural properties suggests that network architecture can be designed for efficient search without controlling the precise arrangement of connections. Specifically, increasing the number of loops substantially decreases search times, pointing to a potential physical mechanism for regulating reaction rates within organelle network structures.
Asunto(s)
Algoritmos , Redes y Vías Metabólicas , Modelos Teóricos , Orgánulos/fisiología , Biología de Sistemas , HumanosRESUMEN
Fer kinase, a protein involved in the regulation of cell-cell adhesion and proliferation, has been shown to be required during invertebrate development and has been implicated in leukemia, gastric cancer, and liver cancer. However, in vivo roles for Fer during vertebrate development have remained elusive. In this study, we bridge the gap between the invertebrate and vertebrate realms by showing that Fer kinase is required during zebrafish embryogenesis for normal hematopoiesis and vascular organization with distinct kinase dependent and independent functions. In situ hybridization, quantitative PCR and fluorescence activated cell sorting (FACS) analyses revealed an increase in both erythrocyte numbers and gene expression patterns as well as a decrease in the organization of vasculature endothelial cells. Furthermore, rescue experiments have shown that the regulation of hematopoietic proliferation is dependent on Fer kinase activity, while vascular organizing events only require Fer in a kinase-independent manner. Our data suggest a model in which separate kinase dependent and independent functions of Fer act in conjunction with Notch activity in a divergent manner for hematopoietic determination and vascular tissue organization.
RESUMEN
Mitochondria are dynamic organelles that undergo constant remodeling through the regulation of two opposing processes, mitochondrial fission and fusion. Although several key regulators and physiological stimuli have been identified to control mitochondrial fission and fusion, the role of mitochondrial morphology in the two processes remains to be determined. To address this knowledge gap, we investigated whether morphological features extracted from time-lapse live-cell images of mitochondria could be used to predict mitochondrial fate. That is, we asked if we could predict whether a mitochondrion is likely to participate in a fission or fusion event based on its current shape and local environment. Using live-cell microscopy, image analysis software, and supervised machine learning, we characterized mitochondrial dynamics with single-organelle resolution to identify features of mitochondria that are predictive of fission and fusion events. A random forest (RF) model was trained to correctly classify mitochondria poised for either fission or fusion based on a series of morphological and positional features for each organelle. Of the features we evaluated, mitochondrial perimeter positively correlated with mitochondria about to undergo a fission event. Similarly mitochondrial solidity (compact shape) positively correlated with mitochondria about to undergo a fusion event. Our results indicate that fission and fusion are positively correlated with mitochondrial morphological features; and therefore, mitochondrial fission and fusion may be influenced by the mechanical properties of mitochondrial membranes.
Asunto(s)
Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , GTP Fosfohidrolasas/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Luminiscentes/metabolismo , Mutación/genéticaRESUMEN
Cancer cells have several hallmarks that define their neoplastic behavior. One is their unabated replicative potential that allows cells to continually proliferate, and thereby contribute to increasing tumor burden. The progression of a cell through the cell cycle is regulated by a series of checkpoints that ensures successful transmission of genetic information, as well as various cellular components, including organelles and protein complexes to the two resulting daughter cells. The mitochondrial reticulum undergoes coordinated changes in shape to correspond with specific stages of the cell cycle, the most dramatic being complete mitochondrial fragmentation prior to cytokinesis. To determine whether mitochondrial fission is a required step to ensure proper mitochondrial segregation into two daughter cells, we investigated the importance of mitochondrial dynamics to cell cycle progression. We found that mitochondrial hyperfusion promotes a defect in cell cycle progression characterized by an inability for cells to exit G2/M. Additionally, extended periods of persistent mitochondrial fusion led to robust caspase-dependent cell death. The cell death signals were coordinated through activation and cleavage of caspase-8, promoting a potent death response. These results demonstrate the importance of mitochondrial dynamics in cell cycle progression, and that inhibiting mitochondrial fission regulators may provide a therapeutic strategy to target the replicative potential of cancer cells.
Asunto(s)
Apoptosis , Caspasas/metabolismo , División Celular , Fase G2 , Mitocondrias/metabolismo , Línea Celular Tumoral , Supervivencia Celular , HumanosRESUMEN
We previously performed an RNA interference (RNAi) screen and found that the knockdown of the catalytically inactive phosphatase, MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein], resulted in potent chemoresistance. Our follow-up studies demonstrated that knockdown of MK-STYX prevents cells from undergoing apoptosis through a block in cytochrome c release, but that MK-STYX does not localize proximal to the molecular machinery currently known to control this process. In an effort to define its molecular mechanism, we utilized an unbiased proteomics approach to identify proteins that interact with MK-STYX. We identified the mitochondrial phosphatase, PTPMT1 (PTP localized to mitochondrion 1), as the most significant and unique interaction partner of MK-STYX. We previously reported that knockdown of PTPMT1, an important component of the cardiolipin biosynthetic pathway, is sufficient to induce apoptosis and increase chemosensitivity. Accordingly, we hypothesized that MK-STYX and PTPMT1 interact and serve opposing functions in mitochondrial-dependent cell death. We confirmed that MK-STYX and PTPMT1 interact in cells and, importantly, found that MK-STYX suppresses PTPMT1 catalytic activity. Furthermore, we found that knockdown of PTPMT1 resensitizes MK-STYX knockdown cells to chemotherapeutics and restores the ability to release cytochrome c. Taken together, our data support a model in which MK-STYX controls apoptosis by negatively regulating PTPMT1. Given the important role of PTPMT1 in the production of cardiolipin and other phospholipids, this raises the possibility that dysregulated mitochondrial lipid metabolism may facilitate chemoresistance.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/fisiología , Mitocondrias/metabolismo , Fosfohidrolasa PTEN/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Células HeLa , Humanos , Metabolismo de los Lípidos/fisiología , Fosfohidrolasa PTEN/genética , Interferencia de ARNRESUMEN
Protein Tyrosine Phosphatase localized to the Mitochondrion 1 (PTPMT1) is a dual specificity phosphatase exclusively localized to the mitochondria, and has recently been shown to be a critical component in the cardiolipin biosynthetic pathway. The downregulation of PTPMT1 in pancreatic beta cells has been shown to increase cellular ATP levels and insulin production, however, the generalized role of PTPMT1 in cancer cells has not been characterized. Here we report that downregulation of PTPMT1 activity is sufficient to induce apoptosis of cancer cells. Additionally, the silencing of PTPMT1 decreases cardiolipin levels in cancer cells, while selectively increasing ATP levels in glycolytic media. Additionally, sublethal downregulation of PTPMT1 synergizes with low doses of paclitaxel to promote cancer cell death. Our data suggest that inhibition of PTPMT1 causes a metabolic crisis in cancer cells that induces cell death, and may be a mechanism by which cancer cells can be sensitized to currently available therapies.
Asunto(s)
Muerte Celular/genética , Neoplasias/genética , Fosfohidrolasa PTEN/genética , Adenosina Trifosfato/metabolismo , Cardiolipinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Células HeLa , Humanos , Mitocondrias/enzimología , Mitocondrias/genética , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosfohidrolasa PTEN/antagonistas & inhibidores , Fosfohidrolasa PTEN/metabolismo , Paclitaxel/administración & dosificaciónRESUMEN
Macroautophagy (autophagy) is a cellular recycling program essential for homeostasis and survival during cytotoxic stress. This process, which has an emerging role in disease etiology and treatment, is executed in four stages through the coordinated action of more than 30 proteins. An effective strategy for studying complicated cellular processes, such as autophagy, involves the construction and analysis of mathematical or computational models. When developed and refined from experimental knowledge, these models can be used to interrogate signaling pathways, formulate novel hypotheses about systems, and make predictions about cell signaling changes induced by specific interventions. Here, we present the development of a computational model describing autophagic vesicle dynamics in a mammalian system. We used time-resolved, live-cell microscopy to measure the synthesis and turnover of autophagic vesicles in single cells. The stochastically simulated model was consistent with data acquired during conditions of both basal and chemically-induced autophagy. The model was tested by genetic modulation of autophagic machinery and found to accurately predict vesicle dynamics observed experimentally. Furthermore, the model generated an unforeseen prediction about vesicle size that is consistent with both published findings and our experimental observations. Taken together, this model is accurate and useful and can serve as the foundation for future efforts aimed at quantitative characterization of autophagy.