Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P₂. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P₂ or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.     

First report of Phytopythium vexans causing the “Avocado sadness” in Michoacan,Mexico

Mexico is the main producer, consumer and exporter of avocado in the world, being Michoacan the main producer state contributing more than 80% of the national production. There are phytopathogens that decimate the production causing the death of the tree. Root samples were collected in avocado trees that showed the characteristic symptomatology of the disease known as avocado sadness, the sampling was carried out in four of the main avocado producing towns, in the state of Michoacan, Mexico. The isolation consisted in sowing root tissue in Petri dishes with V8^®-PARPH culture medium, subsequently they were identified morphologically and for species level it was determined by molecular biology, with the PCR-ITS technique. Pathogenicity tests were performed in triplicate with avocado seedlings with more than six leaves. After 24 hours, the inoculated plants expressed decay in the apical part, after 120 hours the leaves showed yellowing and after 15 days there was a generalized wilt on the stem and leaves, re-isolating the phytopathogen Phytopythium vexans. This study confirms the first report of the oomycete P. vexans affecting avocado trees in the most important producing region of the Mexican Republic.     

MICAL2 fine-tunes Arp2/3 for actin branching

The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

Remodeling of branched actin networks enables cell protrusion and sensing of the environment and is essential for cell motility. Migrating cells such as fibroblasts, immune cells, and metastatic cancer cells rely on actin dynamics to generate pushing, pulling, and squeezing forces to propel themselves. Therefore, studying the processes regulating assembly and disassembly of actin filaments is key to understanding cell locomotion in health and disease. One of the most important catalyzers of actin assembly is the Arp2/3 complex, which drives lamellipodia formation and cell protrusion. Arp2/3-generated actin networks are also important for endocytic trafficking, membrane remodeling during vesicle internalization, cargo sorting, and membrane excision (1). The seven-protein ARP2/3 complex contains two unconventional actin-related proteins (ARP2 and ARP3) and five additional subunits (ARPC1–5). Mammals express two isoforms of three of the subunits (ARP3/ARP3B, ARPC1A/ARPC1B, and ARPC5/ARPC5L), resulting in functional diversity depending on the specific isoforms incorporated into the ARP2/3 complex; however, despite some intriguing roles described in muscle development (2) and platelet function (3), little is known about the biological significance of these isoforms.The nucleation activity of ARP2/3 complex is regulated at multiple levels to ensure that new actin generation is spatially and temporally controlled. Activation is controlled by Wiskott Aldrich Syndrome Protein (WASP)–family proteins, which are themselves part of multi-protein complex machines (4). WASP-family protein complexes detect multiple inputs such as membrane phospholipids, protein–protein interactions, or post-translational modifications, and act as signaling hubs to regulate branched actin nucleation. Other proteins, such as cortactin or coronin, also modulate branch stability in an antagonistic manner (5). ARP2/3 can be post-translationally modified by phosphorylation and interaction with negative regulators, whereas actin itself is regulated by targeted oxidation of methionine residues (6). How these feedback loops that control ARP2/3 activity are coordinated with cell function is an intense area of research.Molecule interacting with CasL (MICAL) proteins have emerged as important mediators of targeted protein oxidation (6). MICAL proteins (MICAL1–3) are flavin adenine dinucleotide–binding monooxygenases capable of oxidizing target proteins (including actin), either directly or through generation of diffusible H₂O₂, which in turn oxidizes proteins in close proximity. Actin oxidation occurs on two methionine residues (Met44 and Met47), resulting in F-actin disassembly and increased cofilin-mediated F-actin severing. Although actin is the best characterized MICAL substrate, there remains the intriguing possibility of the existence of additional targets that regulate cytoskeleton dynamics.In this issue, Galloni, Carra, et al. evaluated the ability of ARP2/3 complexes, containing either ARP3 or the ARP3B isoform (i.e., isocomplexes), to promote actin assembly, and determined isoform-specific differences in their activity and molecular regulation (7). As a model system, the authors used HeLa cells infected with vaccinia virus to study actin branching, given that this virus induces actin tail nucleation in the host cells. They noticed that in cells lacking ARP3, the localization of GFP-ARP3 or GFP-ARP3B to actin tails was comparable, and both isoforms were similarly incorporated into ARP2/3 complexes (Fig. 1). However, the length of the actin tails in ARP3B-expressing cells was shorter than in ARP3-expressing counterparts. Given that ARP3 and ARP3B isocomplexes were equivalent in their ability to induce actin polymerization in vitro, these data pointed to a faster disassembly rate as the potential cause underlying shorter actin tails in ARP3B-expressing cells. Indeed, by tracking photoactivatable actin to study its dynamics, the researchers confirmed that the rate of filament disassembly was faster in ARP3B-expressing cells.Open in a separate windowFigure 1.Vaccinia virus surfs on the outside of the cell, forming an actin tail in the cytoplasm that aids its propulsion. Arp2/3 complex is involved in initiating the branched actin structures and shows slow dissociation from the branches when it is stabilized by the linker protein cortactin. When an Arp2/3 complex containing the ARP3B isoform of ARP3 forms, the dissociation is enhanced, as ARP3B is subject to oxidation by MICAL2, which is recruited to branches by coronin, causing cortactin displacement and rapid branch dissociating leading to shorter actin tails.To identify the molecular basis for the differences between ARP3 and ARP3B, the authors tested a series of ARP3 and ARP3B chimeric proteins, which revealed the importance of ARP3B amino acids 281–418 in mediating the functional differences with ARP3. In particular, Met293 was essential for ARP3B to generate short actin tails. Given that MICAL enzymes promote actin filament disassembly through oxidation of actin Met44 and Met47, Galloni, Carra, et al. decided to investigate the possibility that MICAL-induced oxidation of Met293 in ARP3B inhibits ARP3B activity. Fluorescently tagged MICAL2, but not MICAL1, was recruited to vaccinia-induced actin tails at a position relatively distant from the virus itself, similar to the actin-binding protein coronin (8). Down-regulation of MICAL2, but not MICAL1, increased actin tail stability and suppressed the short actin tail phenotype induced by ARP3B overexpression. Using an antibody raised against oxidized Met293, the researchers confirmed that ARP3B oxidation was reduced following MICAL2 knockdown. Recruitment of MICAL2 to actin tails was dependent on coronin 1C expression, and silencing of coronin 1C resulted in actin filament stabilization and reversal of ARP3B-induced actin tail shortening comparable to MICAL2 knockdown. Thus, coronin 1C recruitment of MICAL2 results in ARP3B oxidation on Met293, leading to dissociation of ARP2/3B isocomplexes and consequent actin networks destabilization.Interestingly, the authors noted that the actin nucleation promoting factor cortactin, which stabilizes ARP2/3-mediated branch points along actin filaments, was required for actin tail destabilization in ARP3B overexpressing cells but was not necessary for localization of coronin 1C or MICAL2 to actin tails. One possibility is that cortactin supports local MICAL2-mediated oxidation of ARP3B at branch points to induce filament de-branching, rather than bulk actin filament depolymerization that would result from direct actin oxidation. Since MICAL proteins are directed to specific cytoskeleton locations by interacting with Myosin 5A (9) and Myosin 15 (10), the consequences of MICAL activity on actin cytoskeleton organization and function may be fine-tuned by specific MICAL subcellular localization and interacting partners.Given that actin binds directly to the catalytic monooxygenase and calponin homology domains of MICAL proteins to increase enzyme activity and promote methionine oxidation, it is not entirely surprising that the actin-related ARP3B protein can be oxidized by MICAL2. However, the location of Met293 in ARP3B is not analogous to the Met44 or Met47 residues of actin, which raises questions regarding the mechanism of ARP3B oxidation by MICAL2. Structural modeling of the MICAL3–actin complex positions the actin loop containing Met44 and Met47 near the enzyme active site (11). ARP3B may interact with MICAL2 differently to bring Met293 close to the active site for direct oxidation, or H₂O₂ produced by MICAL2 might diffuse and oxidize highly concentrated nearby proteins. If this second possibility were true, then it is also possible that additional protein targets (e.g., coronin 1C, cortactin, additional ARP2/3 subunits) might also be oxidized on Met or Cys residues. Since the effects of MICAL1 on actin are counteracted via reduction of the oxidized Met residues by the sulfoxide reductase enzyme SelR (12), it remains to be determined if ARP3B can be similarly reactivated.     

Cells assemble invadopodia-like structures and invade into matrigel in a matrix metalloprotease dependent manner in the circular invasion assay

The ability of tumor cells to invade is one of the hallmarks of the metastatic phenotype. To elucidate the mechanisms by which tumor cells acquire an invasive phenotype, in vitro assays have been developed that mimic the process of cancer cell invasion through basement membrane or in the stroma. We have extended the characterization of the circular invasion assay and found that it provides a simple and amenable system to study cell invasion in matrix in an environment that closely mimics 3D invasion. Furthermore, it allows detailed microscopic analysis of both live and fixed cells during the invasion process. We find that cells invade in a protease dependent manner in this assay and that they assemble focal adhesions and invadopodia that resemble structures visualized in 3D embedded cells. We propose that this is a useful assay for routine and medium throughput analysis of invasion of cancer cells in vitro and the study of cells migrating in a 3D environment.     

Human cytomegalovirus regulates bioactive sphingolipids

Sphingolipids are present in membranes of all eukaryotic cells. Bioactive sphingolipids also function as signaling molecules that regulate cellular processes such as proliferation, migration, and apoptosis. Human cytomegalovirus (HCMV) exploits a variety of cellular signaling pathways to promote its own replication. However, whether HCMV modulates lipid signaling pathways is an essentially unexplored area of research in virus-host cell interactions. In this study, we examined the accumulation of the bioactive sphingolipids and the enzymes responsible for the biosynthesis and degradation of these lipids. HCMV infection results in increased accumulation and activity of sphingosine kinase (SphK), the enzyme that generates sphingosine 1-phosphate (S1P) and dihydrosphingosine 1-phosphate (dhS1P). We also utilized a mass spectrometry approach to generate a sphingolipidomic profile of HCMV-infected cells. We show that HCMV infection results in increased levels of dhS1P and ceramide at 24 h, suggesting an enhancement of de novo sphingolipid synthesis. Subsequently dihydrosphingosine and dhS1P decrease at 48 h consistent with attenuation of de novo sphingolipid synthesis. Finally, we present evidence that de novo sphingolipid synthesis and sphingosine kinase activity directly impact virus gene expression and virus growth. Together, these findings demonstrate that host cell sphingolipids are dynamically regulated upon infection with a herpes virus in a manner that impacts virus replication.     

Identification of the insulin-responsive tyrosine phosphorylation sites on IRSp53

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organises the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia. It had been established earlier that IRSp53 is tyrosine phosphorylated in response to stimulation of the insulin and insulin-related growth factor receptors, but the consequences of tyrosine phosphorylation for IRSp53 function are unknown. Here, we have used a variety of IRSp53 truncation and point mutants to identify insulin-responsive tyrosine phosphorylation sites on IRSp53. We have found that the C-terminal half of IRSp53 (residues 251-521) undergoes tyrosine phosphorylation in response to insulin stimulation of the insulin beta receptor or epidermal growth factor stimulation via the epidermal growth factor receptor, and that the key residue for insulin receptor-mediated phosphorylation is tyrosine 310, located in a region between the N-terminal IRSp53/MIM homology domain (IMD, residue 1-250) and the central SH3 domain (residues 374-438) that is predicted to be natively unstructured. Mutation of tyrosine 310 to phenylalanine or glutamic acid abrogates the phosphorylation in response to insulin stimulation, but not in response to stimulation of the epidermal growth factor receptor. The N-terminal IMD, which mediates dimerisation of IRSp53, is required for efficient tyrosine phosphorylation downstream of either the insulin or epidermal growth factor receptor stimulation, yet does not appear to include a tyrosine-phosphorylated site itself. Thus, we have identified tyrosine 310 as a primary site of tyrosine phosphorylation in response to insulin signalling and we have shown that although IRSp53 is tyrosine phosphorylated in response to epidermal growth factor receptor signalling, tyrosine 310 is not crucial. Furthermore, the tyrosine phosphorylation status does not appear to affect the cell morphology and production of filopod-like structures upon expression of IRSp53.     

Arp2/3 complex activity in filopodia of spreading cells

Background  

Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices.     

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in <Emphasis Type="Italic">Shewanella oneidensis</Emphasis>MR-1

Background  

EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.