scholarly journals Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1

2019 ◽  
Vol 30 (20) ◽  
pp. 2543-2557 ◽  
Author(s):  
Kristi E. Miller ◽  
Wing-Cheong Lo ◽  
Ching-Shan Chou ◽  
Hay-Oak Park
Keyword(s):  
Bound States ◽  
Scaffold Protein ◽  
Cell Polarization ◽  
Temporal Regulation ◽  
Ras Gtpase ◽  
Polarized Secretion ◽  
Associated Proteins ◽  
Guanosine Triphosphatase ◽  
Bud Site

The Cdc42 guanosine triphosphatase (GTPase) plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site is selected in the G1 phase. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet how Rsr1 regulates cell polarization is not fully understood. Here, we show that Rsr1-GDP interacts with the scaffold protein Bem1 in early G1, likely hindering the role of Bem1 in Cdc42 polarization and polarized secretion. Consistent with these in vivo observations, mathematical modeling predicts that Bem1 is unable to promote Cdc42 polarization in early G1 in the presence of Rsr1-GDP. We find that a part of the Bem1 Phox homology domain, which overlaps with a region interacting with the exocyst component Exo70, is necessary for the association of Bem1 with Rsr1-GDP. Overexpression of the GDP-locked Rsr1 interferes with Bem1-dependent Exo70 polarization. We thus propose that Rsr1 functions in spatial and temporal regulation of polarity establishment by associating with distinct polarity factors in its GTP- and GDP-bound states.

scholarly journals Temporal regulation of cell polarity via the interaction of the Ras GTPase Rsr1 and the scaffold protein Bem1

2019 ◽  
Author(s):  
Kristi E. Miller ◽  
Hay-Oak Park
Keyword(s):  
Cell Polarity ◽  
Bound States ◽  
Bound State ◽  
Scaffold Protein ◽  
Cell Polarization ◽  
Temporal Regulation ◽  
Ras Gtpase ◽  
Px Domain ◽  
Associated Proteins ◽  
Bud Site

AbstractEstablishing cell polarity is critical for growth and development of most organisms. The Cdc42 GTPase plays a central role in polarity development in species ranging from yeast to humans. In budding yeast, a specific growth site (i.e. bud site) is selected in the G1 phase, which determines the axis of cell polarization. Rsr1, a Ras GTPase, interacts with Cdc42 and its associated proteins to promote polarized growth at the proper bud site. Yet the mechanism underlying spatial cue-directed cell polarization is not fully understood. Here, we show that Rsr1 associates with Bem1, a scaffold protein, preferentially in its GDP-bound state in early G1. This interaction involves a part of the Bem1 Phox homology (PX) domain, which overlaps with a region previously shown to interact with Exo70, an exocyst component. Furthermore, overexpression of the constitutively GDP-bound Rsr1 interferes with Bem1’s association with Exo70 and inhibits Bem1-dependent Exo70 polarization. We propose that Rsr1 plays a delicate role in coordination of spatial and temporal regulation of polarity establishment via its GTP- and GDP-bound states.

scholarly journals The Rsr1/Bud1 GTPase Interacts with Itself and the Cdc42 GTPase during Bud-Site Selection and Polarity Establishment in Budding Yeast

2010 ◽  
Vol 21 (17) ◽  
pp. 3007-3016 ◽  
Author(s):  
Pil Jung Kang ◽  
Laure Béven ◽  
Seethalakshmi Hariharan ◽  
Hay-Oak Park
Keyword(s):  
Budding Yeast ◽  
Cell Polarization ◽  
Spatial Cues ◽  
Division Site ◽  
Heterotypic Interactions ◽  
Polarity Establishment ◽  
Set Up ◽  
Bud Site

Cell polarization occurs along a single axis that is generally determined in response to spatial cues. In budding yeast, the Rsr1 GTPase and its regulators direct the establishment of cell polarity at the proper cortical location in response to cell type–specific cues. Here we use a combination of in vivo and in vitro approaches to understand how Rsr1 polarization is established. We find that Rsr1 associates with itself in a spatially and temporally controlled manner. The homotypic interaction and localization of Rsr1 to the mother-bud neck and to the subsequent division site are dependent on its GDP-GTP exchange factor Bud5. Analyses of rsr1 mutants suggest that Bud5 recruits Rsr1 to these sites and promotes the homodimer formation. Rsr1 also exhibits heterotypic interaction with the Cdc42 GTPase in vivo. We show that the polybasic region of Rsr1 is necessary for the efficient homotypic and heterotypic interactions, selection of a proper growth site, and polarity establishment. Our findings thus suggest that dimerization of GTPases may be an efficient mechanism to set up cellular asymmetry.

scholarly journals Temporal regulation of morphogenetic events inSaccharomyces cerevisiae

2018 ◽  
Vol 29 (17) ◽  
pp. 2069-2083 ◽  
Author(s):  
Helen Lai ◽  
Jian-Geng Chiou ◽  
Anastasia Zhurikhina ◽  
Trevin R. Zyla ◽  
Denis Tsygankov ◽  
...  
Keyword(s):  
Cell Wall ◽  
Budding Yeast ◽  
Cyclin Dependent Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temporal Regulation ◽  
Bud Emergence ◽  
Polarized Secretion ◽  
Local Cell ◽  
Actin Cables ◽  
Bud Site

Tip growth in fungi involves highly polarized secretion and modification of the cell wall at the growing tip. The genetic requirements for initiating polarized growth are perhaps best understood for the model budding yeast Saccharomyces cerevisiae. Once the cell is committed to enter the cell cycle by activation of G1 cyclin/cyclin-dependent kinase (CDK) complexes, the polarity regulator Cdc42 becomes concentrated at the presumptive bud site, actin cables are oriented toward that site, and septin filaments assemble into a ring around the polarity site. Several minutes later, the bud emerges. Here, we investigated the mechanisms that regulate the timing of these events at the single-cell level. Septin recruitment was delayed relative to polarity establishment, and our findings suggest that a CDK-dependent septin “priming” facilitates septin recruitment by Cdc42. Bud emergence was delayed relative to the initiation of polarized secretion, and our findings suggest that the delay reflects the time needed to weaken the cell wall sufficiently for the cell to bud. Rho1 activation by Rom2 occurred at around the time of bud emergence, perhaps in response to local cell-wall weakening. This report reveals regulatory mechanisms underlying the morphogenetic events in the budding yeast.

scholarly journals Role of Nucleotide Binding in Septin-Septin Interactions and Septin Localization in Saccharomyces cerevisiae

2008 ◽  
Vol 28 (16) ◽  
pp. 5120-5137 ◽  
Author(s):  
Satish Nagaraj ◽  
Ashok Rajendran ◽  
Charles E. Jackson ◽  
Mark S. Longtine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Binding ◽  
Temperature Sensitive ◽  
Associated Proteins ◽  
And Function ◽  
Bud Site ◽  
Two Hybrid

ABSTRACT Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.

scholarly journals A Role for the Rap GTPase YlRsr1 in Cellular Morphogenesis and the Involvement of YlRsr1 and the Ras GTPase YlRas2 in Bud Site Selection in the Dimorphic Yeast Yarrowia lipolytica

2014 ◽  
Vol 13 (5) ◽  
pp. 580-590 ◽  
Author(s):  
Yun-Qing Li ◽  
Min Li ◽  
Xiao-Feng Zhao ◽  
Xiang-Dong Gao
Keyword(s):  
Yarrowia Lipolytica ◽  
Site Selection ◽  
Bound States ◽  
Selection Process ◽  
Content Type ◽  
Yeast Form ◽  
Ras Gtpase ◽  
Cellular Morphogenesis ◽  
Dimorphic Yeast ◽  
Bud Site

ABSTRACT Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for Yl RSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of Yl RSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1's function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Yl ras2 Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2's function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.

Three-Dimensional Subcellular Organization of Hepatocytes in Intact Liver: Simultaneous Visualization of Cytoskeletal and Membrane Markers by Confocal Microscopy

1996 ◽  
Vol 54 ◽  
pp. 288-289
Author(s):  
Greg V. Martin ◽  
Ann L. Hubbard
Keyword(s):  
Three Dimensional ◽  
Integral Membrane Proteins ◽  
Polarized Secretion ◽  
Microscope Images ◽  
Computer Aided ◽  
Intracellular Organelles ◽  
Cytoskeletal Elements ◽  
Simultaneous Visualization

The microtubule (MT) cytoskeleton is necessary for many of the polarized functions of hepatocytes. Among the functions dependent on the MT-based cytoskeleton are polarized secretion of proteins, delivery of endocytosed material to lysosomes, and transcytosis of integral plasma membrane (PM) proteins. Although microtubules have been shown to be crucial to the establishment and maintenance of functional and structural polarization in the hepatocyte, little is known about the architecture of the hepatocyte MT cytoskeleton in vivo, particularly with regard to its relationship to PM domains and membranous organelles. Using an in situ extraction technique that preserves both microtubules and cellular membranes, we have developed a protocol for immunofluorescent co-localization of cytoskeletal elements and integral membrane proteins within 20 µm cryosections of fixed rat liver. Computer-aided 3D reconstruction of multi-spectral confocal microscope images was used to visualize the spatial relationships among the MT cytoskeleton, PM domains and intracellular organelles.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

scholarly journals Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy

2021 ◽  
Vol 22 (8) ◽  
pp. 3995
Author(s):  
Cheong-Yong Yun ◽  
Nahyun Choi ◽  
Jae Un Lee ◽  
Eun Jung Lee ◽  
Ji Young Kim ◽  
...  
Keyword(s):  
Related Factor ◽  
Melanin Pigmentation ◽  
Microtubule Associated Proteins ◽  
Melanocyte Stimulating Hormone ◽  
Associated Proteins ◽  
Autophagy Pathway ◽  
Nrf2 Activator ◽  
Promising Therapeutic Agent

Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.

scholarly journals The Scaffold Protein Cybr Is Required for Cytokine-Modulated Trafficking of Leukocytes In Vivo

2006 ◽  
Vol 26 (14) ◽  
pp. 5249-5258 ◽  
Author(s):  
Vincenzo Coppola ◽  
Colleen A. Barrick ◽  
Sara Bobisse ◽  
Maria Cecilia Rodriguez-Galan ◽  
Michela Pivetta ◽  
...  
Keyword(s):  
Immune System ◽  
Cytotoxic T Lymphocyte ◽  
Leukemia Virus ◽  
Physiological Role ◽  
Scaffold Protein ◽  
Nucleotide Exchange ◽  
Lymphocyte Migration ◽  
And Migration

ABSTRACT Trafficking and cell adhesion are key properties of cells of the immune system. However, the molecular pathways that control these cellular behaviors are still poorly understood. Cybr is a scaffold protein highly expressed in the hematopoietic/immune system whose physiological role is still unknown. In vitro studies have shown it regulates LFA-1, a crucial molecule in lymphocyte attachment and migration. Cybr also binds cytohesin-1, a guanine nucleotide exchange factor for the ARF GTPases, which affects actin cytoskeleton remodeling during cell migration. Here we show that expression of Cybr in vivo is differentially modulated by type 1 cytokines during lymphocyte maturation. In mice, Cybr deficiency negatively affects leukocytes circulating in blood and lymphocytes present in the lymph nodes. Moreover, in a Th1-polarized mouse model, lymphocyte trafficking is impaired by loss of Cybr, and Cybr-deficient mice with aseptic peritonitis have fewer cells than controls present in the peritoneal cavity, as well as fewer leukocytes leaving the bloodstream. Mutant mice injected with Moloney murine sarcoma/leukemia virus develop significantly larger tumors than wild-type mice and have reduced lymph node enlargement, suggesting reduced cytotoxic T-lymphocyte migration. Taken together, these data support a role for Cybr in leukocyte trafficking, especially in response to proinflammatory cytokines in stress conditions.

scholarly journals Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

2016 ◽  
Vol 27 (22) ◽  
pp. 3616-3626 ◽  
Author(s):  
Tanumoy Saha ◽  
Isabel Rathmann ◽  
Abhiyan Viplav ◽  
Sadhana Panzade ◽  
Isabell Begemann ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Growth Dynamics ◽  
Automated Analysis ◽  
Cell Types ◽  
Control Mechanisms ◽  
Spatially Resolved ◽  
Novel Approach ◽  
Associated Proteins

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

Sign in / Sign up

Export Citation Format

Share Document