scholarly journals Small GTPases and BAR domain proteins regulate branched actin to make clathrin and dynamin independent endocytic vesicles

2017 ◽  
Author(s):  
Mugdha Sathe ◽  
Gayatri Muthukrishnan ◽  
James Rae ◽  
Andrea Disanza ◽  
Mukund Thattai ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Early Stage ◽  
Large Fraction ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Vesicle ◽  
Bar Domain ◽  
Bar Domain Proteins ◽  
Molecular Machinery ◽  
Endocytic Vesicles

AbstractNumerous endocytic pathways operate simultaneously at the cell surface. Here we focus on the molecular machinery involved in the generation of endocytic vesicles of the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. This pathway internalises many GPI-anchored proteins and a large fraction of the fluid-phase in different cell types. We developed a real-time TIRF assay using pH-sensitive GFP-GPI to identify nascent CG endocytic sites. The temporal profile of known CG pathway modulators showed that ARF1/GBF1 (GTPase/GEF pair) and CDC42 (RhoGTPase) are recruited sequentially to CG endocytic sites, ∼60s and ∼9s prior to scission. Using a limited RNAi screen, we found several BAR domain proteins affecting CG endocytosis and focused on IRSp53 and PICK1 that have interactions with CDC42 and ARF1 respectively. IRSp53, an I-BAR domain containing protein, was recruited to the plasma membrane at the site of forming CG endocytic vesicles and in its absence, nascent endocytic CLICs, did not form. The requirement for actin polymerization in the CG pathway suggested a role for nucleators of actin polymerization, and ARP2/3 was found enriched at the site of the forming endocytic vesicle. PICK1, a BAR domain containing protein and the ARP2/3 inhibitor is recruited at an early stage along with ARP2/3, but is removed from the endocytic site coincident with CDC42 recruitment and a burst of Factin polymerization. This study provides a spatio-temporal understanding of the molecular machinery necessary to build a CG endocytic vesicle.

scholarly journals Publisher Correction: Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mugdha Sathe ◽  
Gayatri Muthukrishnan ◽  
James Rae ◽  
Andrea Disanza ◽  
Mukund Thattai ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Bar Domain ◽  
Bar Domain Proteins

scholarly journals Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis

2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Mugdha Sathe ◽  
Gayatri Muthukrishnan ◽  
James Rae ◽  
Andrea Disanza ◽  
Mukund Thattai ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Bar Domain ◽  
Bar Domain Proteins

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

scholarly journals Multiple inhibitory ligands induce impaired T-cell immunologic synapse function in chronic lymphocytic leukemia that can be blocked with lenalidomide: establishing a reversible immune evasion mechanism in human cancer

Blood ◽  
2012 ◽  
Vol 120 (7) ◽  
pp. 1412-1421 ◽  
Author(s):  
Alan G. Ramsay ◽  
Andrew J. Clear ◽  
Rewas Fatah ◽  
John G. Gribben
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Immune Evasion ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Small Gtpases ◽  
Lymphocytic Leukemia ◽  
Functional Screening ◽  
Immunologic Synapse

Abstract Cancer immune evasion is an emerging hallmark of disease progression. We have demonstrated previously that impaired actin polymerization at the T-cell immunologic synapse is a global immune dysfunction in chronic lymphocytic leukemia (CLL). Direct contact with tumor cells induces defective actin polarization at the synapse in previously healthy T cells, but the molecules mediating this dysfunction were not known. In the present study, we show via functional screening assays that CD200, CD270, CD274, and CD276 are coopted by CLL cells to induce impaired actin synapse formation in both allogeneic and autologous T cells. We also show that inhibitory ligand–induced impairment of T-cell actin dynamics is a common immunosuppressive strategy used by both hematologic (including lymphoma) and solid carcinoma cells. This immunosuppressive signaling targets T-cell Rho-GTPase activation. Of clinical relevance, the immunomodulatory drug lenalidomide prevented the induction of these defects by down-regulating tumor cell–inhibitory molecule expression. These results using human CLL as a model cancer establish a novel evasion mechanism whereby malignant cells exploit multiple inhibitory ligand signaling to down-regulate small GTPases and lytic synapse function in global T-cell populations. These findings should contribute to the design of immunotherapeutic strategies to reverse T-cell tolerance in cancer.

Influence fast or later: two types of influencers in social networks

2021 ◽  
Author(s):  
Fang Zhou ◽  
Chang Su ◽  
Shuqi Xu ◽  
Linyuan Lü
Keyword(s):  
Final Stage ◽  
Early Stage ◽  
Large Fraction ◽  
Structure And Function ◽  
Spreading Process ◽  
Spreading Effect ◽  
Local Structures ◽  
Different Types ◽  
Small Set ◽  
And Function

Abstract In real-world networks, there usually exist a small set of nodes that play an important role in the structure and function of networks. Those vital nodes can influence most other nodes in the network via a spreading process. While most of the existing works focused on vital nodes that can maximize the spreading size in the final stage, which we call final influencers, recent work proposed the idea of fast influencers, which emphasizes nodes’ spreading capacity at the early stage. Despite the recent surge of efforts in identifying these two types of influencers in networks, there remained limited research on untangling the differences between fast influencers and final influencers. In this paper, we first distinguish the two types of influencers: fast-only influencers and final-only influencers. The former is defined as individuals who can achieve a high spreading effect at the early stage but lose their superiority in the final stage, and the latter are those individuals that fail to exhibit a prominent spreading performance at the early stage but influence a large fraction of nodes at the final stage. Further experiments based on eight empirical datasets, we reveal the key differences between the two types of influencers concerning their spreading capacity and the local structures. We also analyze how network degree assortativity influences the fraction of the proposed two types of influencers. The results demonstrate that with the increase of degree assortativity, the fraction of the fast-only influencers decreases, which indicates that more fast influencers tend to keep their superiority at the final stage. Our study provides insights into the differences and evolution of different types of influencers and has important implications for various empirical applications, such as advertisement marketing, and epidemic suppressing.

Rab22a affects the morphology and function of the endocytic pathway

2001 ◽  
Vol 114 (22) ◽  
pp. 4041-4049 ◽  
Author(s):  
Rosana Mesa ◽  
Cristina Salomón ◽  
Marcelo Roggero ◽  
Philip D. Stahl ◽  
Luis S. Mayorga
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Rab Proteins ◽  
Rab Protein ◽  
Late Endosomes ◽  
And Function ◽  
Negative Mutant

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

scholarly journals FAM92A1 is a BAR domain protein required for mitochondrial ultrastructure and function

2018 ◽  
Vol 218 (1) ◽  
pp. 97-111 ◽  
Author(s):  
Liang Wang ◽  
Ziyi Yan ◽  
Helena Vihinen ◽  
Ove Eriksson ◽  
Weihuan Wang ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
Molecular Mechanisms ◽  
Membrane Curvature ◽  
Mitochondrial Morphology ◽  
Membrane Remodeling ◽  
Mitochondrial Ultrastructure ◽  
Bar Domain ◽  
Bar Domain Proteins ◽  
Domain Protein

Mitochondrial function is closely linked to its dynamic membrane ultrastructure. The mitochondrial inner membrane (MIM) can form extensive membrane invaginations known as cristae, which contain the respiratory chain and ATP synthase for oxidative phosphorylation. The molecular mechanisms regulating mitochondrial ultrastructure remain poorly understood. The Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of diverse cellular processes related to membrane remodeling and dynamics. Whether BAR domain proteins are involved in sculpting membranes in specific submitochondrial compartments is largely unknown. In this study, we report FAM92A1 as a novel BAR domain protein localizes to the matrix side of the MIM. Loss of FAM92A1 caused a severe disruption to mitochondrial morphology and ultrastructure, impairing organelle bioenergetics. Furthermore, FAM92A1 displayed a membrane-remodeling activity in vitro, inducing a high degree of membrane curvature. Collectively, our findings uncover a role for a BAR domain protein as a critical organizer of the mitochondrial ultrastructure that is indispensable for mitochondrial function.

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