Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

Actin filaments (F-actin) have been implicated in various steps of endosomal trafficking, and the length of F-actin is controlled by actin capping proteins, such as CapZ, which is a stable heterodimeric protein complex consisting of a and β subunits. However, the role of these capping proteins in endosomal trafficking remains elusive. Here, we found that CapZ docks to endocytic vesicles via its C-terminal actin-binding motif. CapZ knockout significantly increases the F-actin density around immature early endosomes, and this impedes fusion between these vesicles, manifested by the accumulation of small endocytic vesicles in CapZ-knockout cells. CapZ also recruits several RAB5 effectors, such as Rabaptin-5, to RAB5-positive early endosomes via its N-terminal domain, and this further activates RAB5. Collectively, our results indicate that CapZ regulates endosomal trafficking by controlling actin density around early endosomes and recruiting RAB5 effectors.

Evolutionary mode for the functional preservation of fast-evolving Drosophila telomere capping proteins

DNA end protection is fundamental for the long-term preservation of the genome. In vertebrates the Shelterin protein complex protects telomeric DNA ends, thereby contributing to the maintenance of genome integrity. In the Drosophila genus, this function is thought to be performed by the Terminin complex, an assembly of fast-evolving subunits. Considering that DNA end protection is fundamental for successful genome replication, the accelerated evolution of Terminin subunits is counterintuitive, as conservation is supposed to maintain the assembly and concerted function of the interacting partners. This problem extends over Drosophila telomere biology and provides insight into the evolution of protein assemblies. In order to learn more about the mechanistic details of this phenomenon we have investigated the intra- and interspecies assemblies of Verrocchio and Modigliani, two Terminin subunits using in vitro assays. Based on our results and on homology-based three-dimensional models for Ver and Moi, we conclude that both proteins contain Ob-fold and contribute to the ssDNA binding of the Terminin complex. We propose that the preservation of Ver function is achieved by conservation of specific amino acids responsible for folding or localized in interacting surfaces. We also provide here the first evidence on Moi DNA binding.

First person – Anja Schmidt and Long Li

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Anja Schmidt and Long Li are co-first authors on ‘ Dia- and Rok-dependent enrichment of capping proteins in a cortical region’, published in JCS. Anja conducted the research described in this article while a postdoc in Jörg Großhans's lab at Georg-August-University Göttingen, Germany. She is now a postdoc in the lab of Mark Peifer at Chapel Hill, NC, USA, investigating cell–cell interactions, mechanotransduction and cell–cell junction dynamics. Long is a postdoc in the lab of Jörg Großhans at Philipps-Universität Marburg, Germany, where he focuses on the regulation and interaction between microtubule and actin interaction.

Dia- and Rok-dependent enrichment of capping proteins in a cortical region

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos Rho1 signaling is high between actin caps, i. e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. Here, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that Capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok/MyoII control Capping protein enrichment and support a model that Dia and Rok/MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling.

Molecular Determinants of Filament Capping Proteins Required for the Formation of Functional Flagella in Gram-Negative Bacteria

Bacterial flagella are cell surface protein appendages that are critical for motility and pathogenesis. Flagellar filaments are tubular structures constructed from thousands of copies of the protein flagellin, or FliC, arranged in helical fashion. Individual unfolded FliC subunits traverse the filament pore and are folded and sorted into place with the assistance of the flagellar capping protein complex, an oligomer of the FliD protein. The FliD filament cap is a stool-like structure, with its D2 and D3 domains forming a flat head region, and its D1 domain leg-like structures extending perpendicularly from the head towards the inner core of the filament. Here, using an approach combining bacterial genetics, motility assays, electron microscopy and molecular modeling, we define, in numerous Gram-negative bacteria, which regions of FliD are critical for interaction with FliC subunits and result in the formation of functional flagella. Our data indicate that the D1 domain of FliD is its sole functionally important domain, and that its flexible coiled coil region comprised of helices at its extreme N- and C-termini controls compatibility with the FliC filament. FliD sequences from different bacterial species in the head region are well tolerated. Additionally, head domains can be replaced by small peptides and larger head domains from different species and still produce functional flagella.

Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

The bacterial flagellum is a complex and dynamic nanomachine that propels bacteria through liquids. It consists of a basal body, a hook, and a long filament. The flagellar filament is composed of thousands of copies of the protein flagellin (FliC) arranged helically and ending with a filament cap composed of an oligomer of the protein FliD. The overall structure of the filament core is preserved across bacterial species, while the outer domains exhibit high variability, and in some cases are even completely absent. Flagellar assembly is a complex and energetically costly process triggered by environmental stimuli and, accordingly, highly regulated on transcriptional, translational and post-translational levels. Apart from its role in locomotion, the filament is critically important in several other aspects of bacterial survival, reproduction and pathogenicity, such as adhesion to surfaces, secretion of virulence factors and formation of biofilms. Additionally, due to its ability to provoke potent immune responses, flagellins have a role as adjuvants in vaccine development. In this review, we summarize the latest knowledge on the structure of flagellins, capping proteins and filaments, as well as their regulation and role during the colonization and infection of the host.

Erythroid differentiation in mouse erythroleukemia cells is driven via actin filament-tropomodulin3-tropomyosin networks

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament assembly and organization during terminal ED have revealed essential roles for the pointed-end actin filament capping proteins, tropomodulins (Tmod1 and Tmod3). Additionally, tropomyosin (Tpm) binding to Tmods is a key feature promoting Tmod-mediated actin filament capping. Global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To test a cell autonomous function for Tmod3 and further decipher its biochemical function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability and increased apoptosis. In Mel ds19 cells, both Tpms and actin were preferentially associated with the Triton-X 100 insoluble cytoskeleton during ED, indicating Tpm-coated actin filament assembly during ED. While loss of Tmod3 did not lead to a change in total actin levels, it led to a severe reduction in the proportion of Tpms and actin associated with the Triton-X 100 insoluble cytoskeleton during ED. We conclude that Tmod3-regulation of actin cytoskeleton assembly via Tpms is integral to morphological maturation and cell survival during normal erythroid terminal differentiation.

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants show impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, affecting the morphogenesis of Kupffer’s vesicle (KV). Mutant KVs are smaller and display decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This first in vivo animal study of CARMIL3 reveals its new role for CARMIL3 during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

Structure of an atypical homodimeric actin capping protein from the malaria parasite

Actin capping proteins (CPs) are essential regulators of actin dynamics in all eukaryotes. Their structure and function have been extensively characterized in higher eukaryotes but their role and mechanism of action in apicomplexan parasites remain enigmatic. Here, we present a crystal structure of a unique homodimeric CP from the rodent malaria parasite Plasmodium berghei. In addition, we compare homo- and heterodimeric arrangements of P. berghei CPs (PbCPs) in solution. We complement our findings by describing the regulatory effects of PbCPs on heterologous skeletal muscle α-actin as well as parasite actin. Comprehensive kinetic and steadystate measurements show atypical regulation of actin dynamics; PbCPs facilitate rapid turnover of parasite actin I without affecting the apparent critical concentration. Possibly to rescue actin filament capping in life cycle stages where the CP β-subunit is downregulated, homo- and heterodimeric PbCPs show redundant effects in vitro. However, our data suggest that homodimers may in addition influence actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks the structural elements essential for canonical barbed end interactions, suggesting a novel CP binding mode. These findings facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium and the eligibility of PbCPs as drug targets against malaria.Significance statementMalaria parasites of the genus Plasmodium display a unique form of gliding motility, which depends on an unconventional actomyosin motor. Actin capping proteins (CPs) play a major role in regulating parasite motility. Here, we describe a unique Plasmodium berghei CP (PbCP) system, behaving contradictory to canonical heterodimeric CPs, more suited to regulate the fast dynamics of the parasite actin system. We present the crystal structure of a distinctive homodimeric form of PbCP and extensive biochemical data, describing the atypical behavior of each PbCP form. The PbCP homodimer displays capping even in the absence of canonical conserved structural elements, suggesting a novel actin-CP interaction mode. These distinct structural properties could provide opportunities for drug design against malaria.