scholarly journals Cell3: A new vision for study of the endomembrane system in mammalian cells

2021 ◽  
Author(s):  
Margaritha M. Mysior ◽  
Jeremy C. Simpson
Keyword(s):  
Membrane Trafficking ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Function ◽  
Cultured Cells ◽  
Membrane Traffic ◽  
Endomembrane System ◽  
Cellular Environment ◽  
New Vision ◽  
The Individual

The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues, and present an exciting new opportunity for the study of cell function. In this mini-review we summarise the main methods used to generate 3D cell models, and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool towards deepening our understanding of the endomembrane system in mammalian cells.

scholarly journals Cell3: a new vision for study of the endomembrane system in mammalian cells

2021 ◽  
Vol 41 (12) ◽  
Author(s):  
Margaritha M. Mysior ◽  
Jeremy C. Simpson
Keyword(s):  
Membrane Trafficking ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Function ◽  
Cultured Cells ◽  
Membrane Traffic ◽  
Endomembrane System ◽  
Cellular Environment ◽  
New Vision ◽  
The Individual

Abstract The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues and present an exciting new opportunity for the study of cell function. In this mini-review, we summarize the main methods used to generate 3D cell models and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool toward deepening our understanding of the endomembrane system in mammalian cells.

scholarly journals RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1089 ◽  
Author(s):  
Maeve Long ◽  
Tilen Kranjc ◽  
Margaritha M. Mysior ◽  
Jeremy C. Simpson
Keyword(s):  
Rna Interference ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Family Members ◽  
Membrane Traffic ◽  
Small Gtpases ◽  
Endomembrane System ◽  
Rho Family

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

scholarly journals Methods for protein delivery into cells: from current approaches to future perspectives

2020 ◽  
Vol 48 (2) ◽  
pp. 357-365
Author(s):  
Chalmers Chau ◽  
Paolo Actis ◽  
Eric Hewitt
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cellular Damage ◽  
Future Perspectives ◽  
Chemically Modified ◽  
Recent Developments ◽  
Direct Delivery

The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

scholarly journals New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System

Genes ◽  
2020 ◽  
Vol 11 (8) ◽  
pp. 899 ◽  
Author(s):  
James H. Grissom ◽  
Verónica A. Segarra ◽  
Richard J. Chi
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Budding Yeast ◽  
Endocytic Pathway ◽  
Model Organisms ◽  
Endosomal Trafficking ◽  
Endomembrane System ◽  
Recycling Endosome ◽  
New Findings

Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.

Subcellular organization of snRNPS in mammalian cells

1995 ◽  
Vol 53 ◽  
pp. 772-773
Author(s):  
A. Gregory Matera
Keyword(s):  
Rna Processing ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Spatial Organization ◽  
Protein Complexes ◽  
Nuclear Architecture ◽  
Valuable Insight ◽  
Subcellular Organization ◽  
Protein Components ◽  
The Individual

Our laboratory is interested in aspects of eukaryotic gene expression that may be regulated at the level of nuclear architecture and/or localization. Understanding the spatial organization of genes and gene products within mammalian nuclei will provide valuable insight into how the various metabolic processes such as replication, transcription, processing and transport are orchestrated. In order to begin to address these questions, we have chosen to study the cell biology of RNA processing. Small RNA-protein complexes known as ribonucleoproteins (RNPs) are central factors in numerous RNA processing reactions. Much of what is currently known about the subcellular organization of RNPs is based upon the locations of the protein components of RNPs, not the RNA components. Furthermore, since many RNAs share common protein subunits it is essential to develop probes which are specific to the individual RNAs. We utilize highly specific antisense oligonucleotide probes and fluorescence in situ hybridization (FISH) to ascertain the organization of RNPs and their associations with particular subdomains within mammalian cells.

scholarly journals OCRL1 function in renal epithelial membrane traffic

2010 ◽  
Vol 298 (2) ◽  
pp. F335-F345 ◽  
Author(s):  
Shanshan Cui ◽  
Christopher J. Guerriero ◽  
Christina M. Szalinski ◽  
Carol L. Kinlough ◽  
Rebecca P. Hughey ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cathepsin D ◽  
Cultured Cells ◽  
Membrane Traffic ◽  
Lowe Syndrome ◽  
Marker Protein ◽  
Lysosomal Hydrolase ◽  
Renal Epithelial Cells ◽  
Lipid Phosphatase ◽  
Golgi Network

The X-linked disorder Lowe syndrome arises from mutations in OCRL1, a lipid phosphatase that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2). Most patients with Lowe syndrome develop proteinuria very early in life. PIP2 dynamics are known to modulate numerous steps in membrane trafficking, and it has been proposed that OCRL1 activity regulates the biogenesis or trafficking of the multiligand receptor megalin. To examine this possibility, we investigated the effects of siRNA-mediated OCRL1 knockdown on biosynthetic and postendocytic membrane traffic in canine and human renal epithelial cells. Cells depleted of OCRL1 did not have significantly elevated levels of cellular PIP2 but displayed an increase in actin comets, as previously observed in cultured cells derived from Lowe patients. Using assays to independently quantitate the endocytic trafficking of megalin and of megalin ligands, we could observe no defect in the trafficking or function of megalin upon OCRL1 knockdown. Moreover, apical delivery of a newly synthesized marker protein was unaffected. OCRL1 knockdown did result in a significant increase in secretion of the lysosomal hydrolase cathepsin D, consistent with a role for OCRL1 in membrane trafficking between the trans-Golgi network and endosomes. Together, our studies suggest that OCRL1 does not directly modulate endocytosis or postendocytic membrane traffic and that the renal manifestations observed in Lowe syndrome patients are downstream consequences of the loss of OCRL1 function.

Membrane traffic in cytokinesis

2005 ◽  
Vol 33 (6) ◽  
pp. 1290-1294 ◽  
Author(s):  
J. Matheson ◽  
X. Yu ◽  
A.B. Fielding ◽  
G.W. Gould
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Membrane Traffic ◽  
Protein Composition ◽  
Early Event ◽  
Contractile Ring ◽  
Purse String ◽  
Daughter Cells ◽  
Spatial Coordinates

A crucial facet of mammalian cell division is the separation of two daughter cells by a process known as cytokinesis. An early event in cytokinesis is the formation of an actomyosis contractile ring, which functions like a purse string in the constriction of the forming furrow between the cells. Far less well characterized are the membrane-trafficking steps which deliver new membrane to the cell surface during the plasma membrane expansion known to accompany furrow formation. It is now clearly established that the plasma membrane at the cleavage furrow of mammalian cells has a distinct lipid and protein composition from the rest of the plasma membrane. This may reflect a requirement for both increased surface area during furrowing and for the co-ordinated delivery of intracellular signalling or membrane re-modelling activities to the correct spatial coordinates during cleavage. In this review, we discuss recent work within the area of membrane traffic and cytokinesis.

scholarly journals Itaconate Alters Succinate and Coenzyme A Metabolism via Inhibition of Mitochondrial Complex II and Methylmalonyl-CoA Mutase

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 117
Author(s):  
Thekla Cordes ◽  
Christian M. Metallo
Keyword(s):  
Amino Acid ◽  
Metabolic Pathways ◽  
Mammalian Cells ◽  
Coenzyme A ◽  
Cultured Cells ◽  
Tca Cycle ◽  
Regulatory Function ◽  
Reversible Inhibitor ◽  
Complex Ii ◽  
Branched Chain

Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing and mass spectrometry approaches to explore how itaconate impacts various metabolic pathways in cultured cells. Itaconate is a competitive and reversible inhibitor of Complex II/succinate dehydrogenase (SDH) that alters tricarboxylic acid (TCA) cycle metabolism leading to succinate accumulation. Upon activation with coenzyme A (CoA), itaconyl-CoA inhibits adenosylcobalamin-mediated methylmalonyl-CoA (MUT) activity and, thus, indirectly impacts branched-chain amino acid (BCAA) metabolism and fatty acid diversity. Itaconate, therefore, alters the balance of CoA species in mitochondria through its impacts on TCA, amino acid, vitamin B12, and CoA metabolism. Our results highlight the diverse metabolic pathways regulated by itaconate and provide a roadmap to link these metabolites to potential downstream biological functions.

scholarly journals Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2116
Author(s):  
Xiaoyong Wang ◽  
Lijuan Zhang ◽  
Qi Dai ◽  
Hongzong Si ◽  
Longyun Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Cyclin Dependent Kinase ◽  
Xenograft Mice ◽  
The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

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