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CYRI-A- colocalizes with plasma membrane-associated nascent macropinocytic structures. (See Video 3.) (A–D) Time sequence images of COS-7 cells expressing P16-GFP-CYRI-A or P17-GFP-CYRI-B (cyan) and either mCherry-tagged CLC15 (clathrin light chain 15; A and B), Caveolin-1 (C), or ARF1 (D). Scale bar = 10 µm for full-size image and 5 µm for zooms. (E and F) Time sequence images of live COS-7 cells coexpressing either P16-mCherry-CYRI-A WT or P16-mCherry-CYRI-A RRDD mutant (magenta) and P16-GFP-CYRI-A WT (cyan). (G–L) COS-7 cells coexpressing P16-GFP-CYRI-A (cyan) and two independent PIP3 reporters (magenta), PH-Grp1 (G–I) or PH-Btk (J–L; n = 31 events in 3 cells for Grp1; n = 9 events in 1 cell for Btk). Red line represents the average value. Scale bar = 10 µm for full-size image and 5 µm for zooms. (M and N) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mScarlet-Lck (labeling the plasma membrane; magenta). The time Lck resides on the vesicles before CYRI-A is recruited is quantified in N (n = 48 events in 10 cells). Scale bar = 10 µm for full-size image and 3 µm for zooms. Red line indicates the average value.
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The Scar/WAVE complex drives actin nucleation during cell migration. Interestingly, the same complex is important in forming membrane ruffles during macropinocytosis, a process mediating nutrient uptake and membrane receptor trafficking. Mammalian CYRI-B is a recently described negative regulator of the Scar/WAVE complex by RAC1 sequestration, but...
Citations
... Laboratory strains of Dictyostelium rely on macropinocytosis for the uptake of essential nutrients, such as glucose and amino acids, from liquid media (Hacker et al., 1997;Williams and Kay, 2018;Zhang et al., 2022). In addition, macropinocytosis has been implicated in pathogen infiltration, cell migration, and plasma membrane repair (Le et al., 2021;Moreau et al., 2019;Saeed et al., 2010;Sonder et al., 2021). ...
Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.
... CYRIA acts as a crucial RAC1-binding regulator, influencing the dynamics of lamellipodia and macropinocytic uptake (Machesky 2023). It plays an important role in moderating migration of cancer cells by controlling macropinosome formation and the regulation of integrin internalization (Le et al. 2021). SUGCT is a mitochondrial enzyme involved in synthesizing glutaryl-CoAis. ...
The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.
... Yet, neither the surface coverage nor the thrombus volume of Fam49b −/− samples differed significantly from controls, indicating that thrombus formation is not altered by FAM49b deficiency ( Figure 7B). However, lack of FAM49b was already reported to increase cell migration in other cell types [12], and downregulation of FAM49 was shown to enhance the invasiveness and migration of cancer cells [14,24]. However, the role of FAM49b in platelet migration is unclear. ...
Platelet function at vascular injury sites is tightly regulated through the actin cytoskeleton. The Wiskott–Aldrich syndrome protein-family verprolin-homologous protein (WAVE)-regulatory complex (WRC) activates lamellipodia formation via ARP2/3, initiated by GTP-bound RAC1 interacting with the WRC subunit CYFIP1. The protein FAM49b (Family of Unknown Function 49b), also known as CYRI-B (CYFIP-Related RAC Interactor B), has been found to interact with activated RAC1, leading to the negative regulation of the WRC in mammalian cells. To investigate the role of FAM49b in platelet function, we studied platelet-specific Fam49b−/−-, Cyfip1−/−-, and Cyfip1/Fam49b−/−-mice. Platelet counts and activation of Fam49b−/− mice were comparable to those of control mice. On fully fibrinogen-coated surfaces, Fam49b−/−-platelets spread faster with an increased mean projected cell area than control platelets, whereas Cyfip1/Fam49b−/−-platelets did not form lamellipodia, phenocopying the Cyfip1−/−-platelets. However, Fam49b−/−-platelets often assumed a polarized shape and were more prone to migrate on fibrinogen-coated surfaces. On 2D structured micropatterns, however, Fam49b−/−-platelets displayed reduced spreading, whereas spreading of Cyfip1−/−- and Cyfip1/Fam49b−/−-platelets was enhanced. In summary, FAM49b contributes to the regulation of morphology and migration of spread platelets, but to exert its inhibitory effect on actin polymerization, the functional WAVE complex must be present.
... 8 In mammals, it plays an important role in nutrient acquisition, immune surveillance, synaptic activity regulation, and cell migration. [9][10][11] Macropinocytosis is hijacked by pathogens, such as viruses and bacteria, to invade cells 12 and is exploited by cancer cells to promote their growth and proliferation in the nutrient-poor tumor microenvironment by scavenging extracellular proteins and fatty acids. [13][14][15][16][17][18] Macropinocytic activity is regulated by diverse environmental factors. ...
... Concentrating on genes that consistently produced a gain-of-function phenotype when depleted by three independent sgRNAs, we identified the Rac-GTP-binding protein CYRI-B as a prominent negative regulator of CEACAM3-driven signaling events. CYRI-B sequesters GTP-bound Rac, thereby limiting Rac-driven downstream processes such as activation of PAK kinases and lamellipodia formation (Fort et al., 2018;Shang et al., 2018;Yuki et al., 2019;Le et al., 2021). We found that knockout of CYRI-B allowed increased phagocytosis of CEACAM3-binding bacteria, which was reverted by re-expression of wild-type, but not Rac-binding-deficient, CYRI-B. ...
Opsonin-independent phagocytosis mediated by human carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3) has evolved to control a subset of human-restricted bacterial pathogens. CEACAM3 engagement triggers rapid GTP-loading of the small GTPase Rac as a master regulator of cytoskeletal rearrangements and lamellipodia-driven internalization. To identify components of the CEACAM3-initiated signaling cascade, we performed a genome-wide CRISPR/Cas9-based screen in human myeloid cells. Following infection with fluorescently labeled bacteria, cells exhibiting elevated phagocytosis (gain-of-function) as well as cells showing reduced phagocytosis (loss-of-function) were sorted and enrichment of individual single-guide RNAs (sgRNAs) was determined by next generation sequencing. Concentrating on genes whose targeting by three distinct sgRNAs consistently resulted in a gain-of-function phenotype, we identified the Rac-GTP-sequestering protein CYRI-B as a negative regulator of CEACAM3-mediated phagocytosis. Clonal HL-60 cell lines with CYRI-B knockout showed enhanced CEACAM3-downstream signaling, such as Rac GTP loading and phosphorylation of PAK kinases, leading to increased phagocytosis of bacteria. Complementation of the CYRI-B knockout cells reverted the knockout phenotype. Our results unravel components of CEACAM3-initiated opsonin-independent phagocytosis on a genome-wide level and highlight CYRI-B as a negative regulator of CEACAM3-initiated signaling in myeloid cells.
... Additionally, known localization-based Rac and Cdc42 sensors were included. Plus, Bem1 and Bem3, a recommendation from the Laan laboratory for Cdc42 binding (Laan et al., 2015), and CYRI-A, which was reported by Machesky and colleagues to be a Rac-binding protein (Le et al., 2021) were tested for their potential as sensors. ...
... Although CYRI-A localized in the nucleus, it was not enriched when the active Rac1 is overexpressed. This might be explained by the putative myristoylation of CYRI-A (Le et al., 2021), which might be required for binding of Rac at the plasma membrane but cannot support binding in the nucleus. Next, the rapamycin system was used to activate endogenous Rac through the recruitment of TIAM to the plasma membrane, to measure relocation efficiency of the Rac sensor candidates (Fig. 6A,B; Fig. S4B). ...
... CYRI-A is not part of the WAVE complex but it can disrupt the Rac1-WAVE regulatory complex. CYRI-A has been shown to indicate Rac1 activity by its localization (Le et al., 2021), which confirms the findings of our study. Pak1, Pak3, Pak4, PAR6A and TNK2 are kinases (Bishop and Hall, 2000;Garrard et al., 2003), N-WASP, WASp, IQGAP1, p67phox, IRSp53, CDD42EPI and Bem1 are The plot at the right shows the effect size relative to the mScarlet-I control. ...
Rac (herein referring to the Rac family) and Cdc42 are Rho GTPases that regulate the formation of lamellipoda and filopodia, and are therefore crucial in processes such as cell migration. Relocation-based biosensors for Rac and Cdc42 have not been characterized well in terms of their specificity or affinity. In this study, we identify relocation sensor candidates for both Rac and Cdc42. We compared their (1) ability to bind the constitutively active Rho GTPases, (2) specificity for Rac and Cdc42, and (3) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1, we found a sensor candidate with low relocation efficiency. For Cdc42, we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity at assembling invadopodia. Moreover, we tested several fluorescent proteins and HaloTag for their influence on the recruitment efficiency of the Rho location sensor, to find optimal conditions for a multiplexing experiment. This characterization and optimization of relocation sensors will broaden their application and acceptance.
... WAVE1, but not WAVE2, is selectively required for CDR formation in PDGF-stimulated fibroblasts. The mechanisms that distinguish WAVE-mediated actin polymerization that produces a CDR versus a membrane ruffle is not clear, but the localization of regulatory proteins like Leep1 [115] and CYRI-A [116] appears to be important (discussed below). Macropinocytosis also requires the action of CARMIL1 and CARMIL2, which are recruited to macropinocytic cups [117,118]. ...
... The termination of Rac1 signaling during macropinocytic cup closure ( Figure 5) could involve the recruitment of the Scar/WAVE antagonist CYRI-A during macropinosome formation [116]. In Cos-7 cells, CYRI-A was recruited to macropinosomes in a PI3K-dependent manner, where it bound to Rac1 and inhibited Scar/ WAVE1 by Rac sequestration. ...
... Binding of microtubule motor proteins by Arf6/JIP3 also promotes sealing; the mechanisms are unclear but have been suggested to involve transit of cargo to the macropinocytic cup. localized in the macropinocytic cup, and it may act both before and after cup closure [116]. Termination of Arf6 signaling may also be required, as Rab35 recruitment of the Arf6 GAP ACAP2 regulates cup closure in both macropinocytosis and phagocytosis [140,141]. ...
Macropinocytosis is defined as an actin-dependent but coat- and dynamin-independent endocytic uptake process, which generates large intracellular vesicles (macropinosomes) containing a non-selective sampling of extracellular fluid. Macropinocytosis provides an important mechanism of immune surveillance by dendritic cells and macrophages, but also serves as an essential nutrient uptake pathway for unicellular organisms and tumor cells. This review examines the cell biological mechanisms that drive macropinocytosis, as well as the complex signaling pathways - GTPases, lipid and protein kinases and phosphatases, and actin regulatory proteins - that regulate macropinosome formation, internalization, and disposition.
... Protrusion -Initiation of macropinocytosis begins with the protrusion of a membrane ruffle driven by WRC and Arp2/3-mediated actin assembly [10][11][12][13][14][15]. Protrusions can take the form of laterally or dorsally extending ruffles and in macrophages, they can be underpinned by actin tent-pole-like structures that aid closure [6,16]. ...
... Closure of the cup -Cup closure involves the fusion of the extended membrane into a vesicle or series of vesicles that are then taken into the cell. This requires remodelling and disassembly of the actin structures and is accompanied a transient increase in CYRI-A followed by a decrease in RAC1 signal at the membrane of the nascent macropinosome [14,17]. Both losses of CYRI and hyperactivation of RAC1 do not prevent the initial extension of ruffles, but prevent subsequent resolution or engulfment of macropinocytic cups, showing the need for dynamic actin remodelling. ...
... Another possibility is that RAC1 or other signalling molecules are similarly repurposed to macropinosomes and away from migratory actin structures. In mammalian cells, it isn't clear whether macropinocytosis and migration play opposing roles, but deletion of the CYRI protein caused a reciprocal inhibition of macropinocytosis and enhancement of migration [1,14], suggesting that at least in some cell types this might hold true. ...
Cells use actin-based protrusions not only to migrate, but also to sample their environment and take up liquids and particles, including nutrients, antigens and pathogens. Lamellipodia are sheet-like actin-based protrusions involved in sensing the substratum and directing cell migration. Related structures, macropinocytic cups, arise from lamellipodia ruffles and can take in large gulps of the surrounding medium. How cells regulate the balance between using lamellipodia for migration and macropinocytosis is not yet well understood. We recently identified CYRI proteins as RAC1-binding regulators of the dynamics of lamellipodia and macropinocytic events. This review discusses recent advances in our understanding of how cells regulate the balance between eating and walking by repurposing their actin cytoskeletons in response to environmental cues.
... Additionally, known localizationbased Rac and Cdc42 sensors were included. Plus, Bem1 and Bem3 a recommendation by the Laan lab for Cdc42 binding (Laan et al., 2015) and CYRI-A which was reported by Machesky and colleagues as a Rac binding protein (Le et al., 2021). ...
... Although CYRI-A localizes in the nucleus, it is not enriched when the active Rac1 is overexpressed. This may be explained by the putative myristoylation of CYRI-A (Le et al., 2021) which may be absent in the nuclear pool. Next, The rapamycin system was applied to activate endogenous Rac by the recruitment of TIAM to the plasma membrane, to measure relocation efficiency of the Rac sensor candidates (Fig 6 A,B, S6B). ...
... CYRI-A is not part of the WAVE complex but it can disrupt the Rac1-WAVE regulatory complex. CYRI-A has been shown to indicate Rac1 activity by its localization (Le et al., 2021), which confirms the findings of our study. Pak1, Pak3, Pak4, PAR6A and TNK2 are kinases (Bishop and Hall, 2000;Garrard et al., 2003), N-WASP, WASp, IQGAP1, p67phox, BAIAP2, CDD42EPI and Bem1 are scaffold proteins (Bishop and Hall, 2000;Burbelo et al., 1999;Laan et al., 2015). ...
Rac and Cdc42 are Rho GTPases which regulate the formation of lamellipoda and filopodia and are therefore crucial in cellular processes such as cell migration. Both Rho GTPases are active at the leading edge of migrating cells. However, it is unclear how their signaling patterns differ from each other and what their specific roles are. To live image their signaling with high spatial and temporal resolution, location-based biosensors (relocating to the native location of the endogenous active Rho GTPase) are attractive probes. Until now, Rac and Cdc42 relocation sensors have not been characterized well, especially in terms of their specificity for the Rho GTPases. In this study, we identify a number of relocation sensor candidates for either Rac or Cdc42. We compared their (i) ability to bind the constitutively active Rho GTPases, (ii) specificity for Rac and Cdc42 and (iii) relocation efficiency in cell-based assays. Subsequently, the relocation efficiency was improved by a multi-domain approach. For Rac1 we found a sensor candidate with low relocation efficiency. For Cdc42 we found several sensors with sufficient relocation efficiency and specificity. These optimized sensors enable the wider application of Rho GTPase relocation sensors, which was showcased by the detection of local endogenous Cdc42 activity in a spreading endothelial cell and a multiplexing experiment with Rho and a Cdc42 relocation sensors. Moreover, we tested several fluorescent proteins and a HaloTag for their influence on the recruitment efficiency of the Rho location senor, to find optimal conditions for the multiplexing experiment. The characterization and optimization of relocation sensors will broaden their application and acceptance in the field of biosensors.
... However, instead of pushing in the plane of the front-rear axis of the cell, local actin polymerisation pushes plasma membrane sheets forward or upward, to form cup-like structures that resolve into macropinocytic vesicles taken into the cell. Cells use macropinocytosis not only to uptake nutrients but also to traffic and organize different membrane receptors, such as integrins, to modulate their adhesion and invasion in cancer (Le et al., 2021). ...
... In our recent paper published in the Journal of Cell Biology (Le et al., 2021), we utilised an image-based internalisation assay, to show that cells lacking CYFIP-related Rac1 Interactor (CYRI) proteins displayed a reduced macropinocytic uptake of dextran 70 kDa. ...
... We believe this is something that is still missing in the current literature, where there are a lot of resources for image analysis, but many are not necessarily accessible or presented in understandable terms for everyone, particularly beginners. The image analysis pipeline presented here is also suitable for analysing any other intracellular signal, including but not limited to integrin internalisation (Le et al., 2021), transferrin, and other endocytic processes. ...
Macropinocytosis is an evolutionarily conserved process, which is characterized by the formation of membrane ruffles and the uptake of extracellular fluid. We recently demonstrated a role for CYFIP-related Rac1 Interactor (CYRI) proteins in macropinocytosis. High-molecular weight dextran (70kDa or higher) has generally been used as a marker for macropinocytosis because it is too large to fit in smaller endocytic vesicles, such as those of clathrin or caveolin-mediated endocytosis. Through the use of an image-based dextran uptake assay, we showed that cells lacking CYRI proteins internalise less dextran compared to their wild-type counterparts. Here, we will describe a step-by-step experimentation procedure to detect internalised dextran in cultured cells, and an image pipeline to analyse the acquired images, using the open-access software ImageJ/Fiji. This protocol is detailed yet simple and easily adaptable to different treatment conditions, and the analysis can also be automated for improved processing speed.
