scholarly journals Comprehensive knockout analysis of the Rab family GTPases in epithelial cells

2019 ◽  
Vol 218 (6) ◽  
pp. 2035-2050 ◽  
Author(s):  
Yuta Homma ◽  
Riko Kinoshita ◽  
Yoshihiko Kuchitsu ◽  
Paulina S. Wawro ◽  
Soujiro Marubashi ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Mdck Cells ◽  
Small Gtpases ◽  
Intracellular Membrane ◽  
General Requirement ◽  
Functional Compensation ◽  
Comprehensive Collection ◽  
Extracellular Matrix Components ◽  
Rab Family ◽  
Matrix Components

The Rab family of small GTPases comprises the largest number of proteins (∼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.

scholarly journals Human Rab small GTPase- and class V myosin-mediated membrane tethering in a chemically defined reconstitution system

2017 ◽  
Author(s):  
Motoki Inoshita ◽  
Joji Mima
Keyword(s):  
Lipid Bilayers ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Rab Proteins ◽  
Dependent Manner ◽  
Class V ◽  
Rab Family ◽  
Membrane Tethering

AbstractMembrane tethering is a fundamental process essential for compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, have been reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes still remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on apposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane tethering reactions.

scholarly journals Homotypic and heterotypic trans-assembly of human Rab-family small GTPases in reconstituted membrane tethering

2019 ◽  
Author(s):  
Kazuya Segawa ◽  
Naoki Tamura ◽  
Joji Mima
Keyword(s):  
Membrane Trafficking ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Physical Contact ◽  
Molar Ratios ◽  
Membrane Surfaces ◽  
Subcellular Membrane ◽  
Rab Family ◽  
Membrane Tethering ◽  
Protein Components

AbstractMembrane tethering is a highly regulated event that occurs during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, 3a, 4a, 5a, 6a, 7a, 9a, 11a, 27a, and 33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly in a chemically defined reconstitution system. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g., Rab-to-lipid molar ratios of 1:100-1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab-to-lipid ratio of 1:3,000. Comprehensive reconstitution studies further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a, in particular, synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. Thus, our findings establish that, in the physiological context, homotypic and heterotypic trans-assembly of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.

Rab proteins of the endoplasmic reticulum: functions and interactors

2012 ◽  
Vol 40 (6) ◽  
pp. 1426-1432 ◽  
Author(s):  
Carolina Ortiz Sandoval ◽  
Thomas Simmen
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Complex ◽  
Membrane Trafficking ◽  
Small Gtpases ◽  
Rab Proteins ◽  
Retrograde Trafficking ◽  
Rab Family ◽  
Intermediate Compartment

Whereas most of what we know today about the Ras-related small GTPases of the Rab family stems from observations made on Golgi complex, endosome and plasma membrane trafficking, a subset of Rabs localizes in part or predominantly to the ER (endoplasmic reticulum). Here, Rabs such as Rab1, Rab2, Rab6 and Rab33 can regulate the anterograde and retrograde trafficking of vesicles between the Golgi complex, the ERGIC (ER–Golgi intermediate compartment) and the ER itself. However, among the ER-associated Rabs, some Rabs appear to perform roles not directly related to trafficking: these Rabs (e.g. Rab32 or Rab24) could aid proteins of the atlastin and reticulon families in determining the extent and direction of ER tubulation. In so doing, these Rabs regulate not only ER contacts with other organelles such as mitochondria, but also the formation of autophagosomes.

scholarly journals Rab GTPases: Switching to Human Diseases

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 909 ◽  
Author(s):  
Noemi Antonella Guadagno ◽  
Cinzia Progida
Keyword(s):  
Membrane Trafficking ◽  
Intracellular Transport ◽  
Small Gtpases ◽  
Regulatory Proteins ◽  
Human Diseases ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Intracellular Membrane ◽  
And Migration ◽  
And Control

Rab proteins compose the largest family of small GTPases and control the different steps of intracellular membrane traffic. More recently, they have been shown to also regulate cell signaling, division, survival, and migration. The regulation of these processes generally occurs through recruitment of effectors and regulatory proteins, which control the association of Rab proteins to membranes and their activation state. Alterations in Rab proteins and their effectors are associated with multiple human diseases, including neurodegeneration, cancer, and infections. This review provides an overview of how the dysregulation of Rab-mediated functions and membrane trafficking contributes to these disorders. Understanding the altered dynamics of Rabs and intracellular transport defects might thus shed new light on potential therapeutic strategies.

scholarly journals Anti-Fibrotic Activity of an Antimicrobial Peptide in a Drosophila Model

2021 ◽  
pp. 1-15
Author(s):  
Dilan Khalili ◽  
Christina Kalcher ◽  
Stefan Baumgartner ◽  
Ulrich Theopold
Keyword(s):  
Antimicrobial Peptide ◽  
Cell Polarity ◽  
Active Form ◽  
Ectopic Expression ◽  
Secretory Activity ◽  
Ras Oncogene ◽  
Apicobasal Polarity ◽  
Pathological Conditions ◽  
Extracellular Matrix Components ◽  
Matrix Components

Fibrotic lesions accompany several pathological conditions, including tumors. We show that expression of a dominant-active form of the Ras oncogene in <i>Drosophila</i> salivary glands (SGs) leads to redistribution of components of the basement membrane (BM) and fibrotic lesions. Similar to several types of mammalian fibrosis, the disturbed BM attracts clot components, including insect transglutaminase and phenoloxidase. SG epithelial cells show reduced apicobasal polarity accompanied by a loss of secretory activity. Both the fibrotic lesions and the reduced cell polarity are alleviated by ectopic expression of the antimicrobial peptide drosomycin (Drs), which also restores the secretory activity of the SGs. In addition to extracellular matrix components, both Drs and F-actin localize to fibrotic lesions.

scholarly journals Interactions of Lipid Droplets with the Intracellular Transport Machinery

2021 ◽  
Vol 22 (5) ◽  
pp. 2776
Author(s):  
Selma Yilmaz Dejgaard ◽  
John F. Presley
Keyword(s):  
Membrane Trafficking ◽  
Lipid Droplets ◽  
Intracellular Transport ◽  
Neutral Lipids ◽  
Small Gtpases ◽  
Lipid Phase ◽  
Intracellular Organelles ◽  
And Function ◽  
Lipid Phase Separation ◽  
Endocytic Pathways

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

scholarly journals Small GTPases of the Rab and Arf Families: Key Regulators of Intracellular Trafficking in Neurodegeneration

2021 ◽  
Vol 22 (9) ◽  
pp. 4425
Author(s):  
Alazne Arrazola Arrazola Sastre ◽  
Miriam Luque Luque Montoro ◽  
Hadriano M. Lacerda ◽  
Francisco Llavero ◽  
José L. Zugaza
Keyword(s):  
Membrane Trafficking ◽  
Neurodegenerative Disorders ◽  
Synaptic Vesicles ◽  
Intracellular Trafficking ◽  
Small Gtpases ◽  
Constant Flow ◽  
Parkinson’S Diseases ◽  
The Central Nervous System ◽  
Arf Gtpases

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration.

scholarly journals Age related extracellular matrix and interstitial cell phenotype in pulmonary valves

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shaohua Wu ◽  
Vikas Kumar ◽  
Peng Xiao ◽  
Mitchell Kuss ◽  
Jung Yul Lim ◽  
...  
Keyword(s):  
Mechanical Properties ◽  
Extracellular Matrix ◽  
Heart Valve ◽  
Cell Phenotype ◽  
Primary Concern ◽  
Ross Operation ◽  
Age Related ◽  
Cardiovascular Morbidity And Mortality ◽  
Extracellular Matrix Components ◽  
Matrix Components

AbstractHeart valve disease is a common manifestation of cardiovascular disease and is a significant cause of cardiovascular morbidity and mortality worldwide. The pulmonary valve (PV) is of primary concern because of its involvement in common congenital heart defects, and the PV is usually the site for prosthetic replacement following a Ross operation. Although effects of age on valve matrix components and mechanical properties for aortic and mitral valves have been studied, very little is known about the age-related alterations that occur in the PV. In this study, we isolated PV leaflets from porcine hearts in different age groups (~ 4–6 months, denoted as young versus ~ 2 years, denoted as adult) and studied the effects of age on PV leaflet thickness, extracellular matrix components, and mechanical properties. We also conducted proteomics and RNA sequencing to investigate the global changes of PV leaflets and passage zero PV interstitial cells in their protein and gene levels. We found that the size, thickness, elastic modulus, and ultimate stress in both the radial and circumferential directions and the collagen of PV leaflets increased from young to adult age, while the ultimate strain and amount of glycosaminoglycans decreased when age increased. Young and adult PV had both similar and distinct protein and gene expression patterns that are related to their inherent physiological properties. These findings are important for us to better understand the physiological microenvironments of PV leaflet and valve cells for correctively engineering age-specific heart valve tissues.

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