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Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development.

ABSTRACT: Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.

SUBMITTER: Chin S

PROVIDER: S-EPMC8364238 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Actin polymerization drives polar growth in Arabidopsis root hair cells.

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Abi is required for modulation and stability but not localization or activation of the SCAR/WAVE complex.

Project description:The SCAR/WAVE complex drives actin-based protrusion, cell migration, and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signaling to SCAR/WAVE complex localization and activation through its polyproline C-terminal tail. We generated a deletion series of the Dictyostelium discoideum Abi to investigate its exact role in regulation of the SCAR complex and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N terminus of Abi or the C-terminal polyproline tail conferred no detectable defect in complex recruitment to the leading edge or the formation of pseudopods. A fragment containing approximately 20% Abi--and none of the sites that couple to known signaling pathways--allowed the SCAR complex to function with normal localization and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by the inappropriate activation of SCAR. This demonstrates, unexpectedly, that Abi does not mediate the SCAR complex's ability to make pseudopods, beyond its role in complex stability. Instead, we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms.

| S-EPMC3837927 | biostudies-literature

The Scar/WAVE complex drives normal actin protrusions without the VCA domain, but proline-rich domains are required

Project description:The Scar/WAVE regulatory complex (WRC) is the main catalyst of pseudopod and lamellipodium formation during cell migration. Its actin nucleation activity has been attributed to its ability to activate the Arp2/3 complex through the VCA domain of Scar/WAVE. Here we show in both B16-F1 mouse melanoma cells and Dictyostelium discoideum cells that the WRC with its VCA domain deleted can still induce the formation of morphologically normal actin protrusions. Its function is lost only after further deletion of Scar/WAVE’s (in Dictyostelium cells) or both Abi and WAVE’s (in B16-F1 cells) proline-rich domains. Moreover, mass spectrometry analysis identified BAIAP2 and Vasp (both promotors of actin polymerization) as specific interactors of WRC polyproline domains. Thus we conclude that proline-rich domains play a central role in actin nucleation by concentrating other effectors needed to form actin protrusions. Together, these findings suggest a new mechanism for WRC action independent of the VCA-Arp2/3 interaction.

2024-08-30 | PXD037123 | Pride

Spatial and temporal relationships between actin-filament nucleation, capping, and disassembly.

Project description:BackgroundThe leading actin network in motile cells is composed of two compartments, the lamellipod and the lamellum. Construction of the lamellipod requires a set of conserved proteins that form a biochemical cycle. The timing of this cycle and the roles of its components in determining actin network architecture in vivo, however, are not well understood.ResultsWe performed fluorescent speckle microscopy on spreading Drosophila S2 cells by using labeled derivatives of actin, the Arp2/3 complex, capping protein, and tropomyosin. We find that capping protein and the Arp2/3 complex both incorporate at the cell edge but that capping protein dissociates after covering less than half the width of the lamellipod, whereas the Arp2/3 complex dissociates after crossing two thirds of the lamellipod. The lamellipodial actin network itself persists long after the loss of the Arp2/3 complex. Depletion of capping protein by RNAi results in the displacement of the Arp2/3 complex and disappearance of the lamellipod. In contrast, depletion of cofilin, slingshot, twinfilin, and tropomyosin, all factors that control the stability of actin filaments, dramatically expanded the lamellipod at the expense of the lamellum.ConclusionsThe Arp2/3 complex is incorporated into the lamellipodial network at the cell edge but debranches well before the lamellipodial network itself is disassembled. Capping protein is required for the formation of a lamellipodial network but dissociates from the network precisely when filament disassembly is first detected. Cofilin, twinfilin, and tropomyosin appear to play no role in lamellipodial network assembly but function to limit its size.

| S-EPMC3077992 | biostudies-literature

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