Structural basis for subversion of host cell actin cytoskeleton during Salmonella infection.

Secreted bacterial type III secretion system (T3SS) proteins are essential for successful infection by many human pathogens. Both T3SS translocator SipC and effector SipA are critical for Salmonella infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. Here, using cryo-electron microscopy, we show that SipA binds along the F-actin grooves with a unique binding pattern. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through closure of the "back door" of adenosine 5'-triphosphate pocket. We also show that SipC enhances the binding of SipA to F-actin, thus demonstrating that a sequential presence of T3SS proteins in host cells is associated with a sequence of infection events-starting with actin nucleation, filament growth, and stabilization. Together, our data explain the coordinated interplay of a precisely tuned and highly effective mechanism during Salmonella infection and provide a blueprint for interfering with Salmonella effectors acting on actin.

Epithelial restitution in 3D - Revealing biomechanical and physiochemical dynamics in intestinal organoids via fs laser nanosurgery.

Intestinal organoids represent a three-dimensional cell culture system mimicking the mammalian intestine. The application of single-cell ablation for defined wounding via a femtosecond laser system within the crypt base allowed us to study cell dynamics during epithelial restitution. Neighboring cells formed a contractile actin ring encircling the damaged cell, changed the cellular aspect ratio, and immediately closed the barrier. Using traction force microscopy, we observed major forces at the ablation site and additional forces on the crypt sides. Inhibitors of the actomyosin-based mobility of the cells led to the failure of restoring the barrier. Close to the ablation site, high-frequency calcium flickering and propagation of calcium waves occured that synchronized with the contraction of the epithelial layer. We observed an increased signal and nuclear translocation of YAP-1. In conclusion, our approach enabled, for the first time, to unveil the intricacies of epithelial restitution beyond in vivo models by employing precise laser-induced damage in colonoids.

Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation.

BACKGROUND: Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. METHODS: Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. RESULTS: Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. CONCLUSIONS: Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

Convergence of Ras- and Rac-regulated formin pathways is pivotal for phagosome formation and particle uptake in Dictyostelium.

Macroendocytosis comprising phagocytosis and macropinocytosis is an actin-driven process regulated by small GTPases that depend on the dynamic reorganization of the membrane that protrudes and internalizes extracellular material by cup-shaped structures. To effectively capture, enwrap, and internalize their targets, these cups are arranged into a peripheral ring or ruffle of protruding actin sheets emerging from an actin-rich, nonprotrusive zone at its base. Despite extensive knowledge of the mechanism driving actin assembly of the branched network at the protrusive cup edge, which is initiated by the actin-related protein (Arp) 2/3 complex downstream of Rac signaling, our understanding of actin assembly in the base is still incomplete. In the Dictyostelium model system, the Ras-regulated formin ForG was previously shown to specifically contribute to actin assembly at the cup base. Loss of ForG is associated with a strongly impaired macroendocytosis and a 50% reduction in F-actin content at the base of phagocytic cups, in turn indicating the presence of additional factors that specifically contribute to actin formation at the base. Here, we show that ForG synergizes with the Rac-regulated formin ForB to form the bulk of linear filaments at the cup base. Consistently, combined loss of both formins virtually abolishes cup formation and leads to severe defects of macroendocytosis, emphasizing the relevance of converging Ras- and Rac-regulated formin pathways in assembly of linear filaments in the cup base, which apparently provide mechanical support to the entire structure. Remarkably, we finally show that active ForB, unlike ForG, additionally drives phagosome rocketing to aid particle internalization.

ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning.

Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.

Ena/VASP clustering at microspike tips involves lamellipodin but not I-BAR proteins, and absolutely requires unconventional myosin-X.

Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive 2D-cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but the molecular mechanisms driving their formation and their potential functional relevance have remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi is essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Interestingly, and despite the invariable distribution of other relevant marker proteins, the width of lamellipodia in MyoX-KO mutants was significantly reduced as compared with B16-F1 control, suggesting that microspikes contribute to lamellipodium stability. Consistently, MyoX removal caused marked defects in protrusion and random 2D-cell migration. Strikingly, Ena/VASP-deficiency also uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation.

Tension-dependent RHGF-1 recruitment to stress fibers drives robust spermathecal tissue contraction.

Contractile epithelial tubes are found in various organs, such as lung airways and blood capillaries. Their ability to sense luminal pressure and respond with adequate contractility is essential for their physiology, and its mis-regulation results in diseases such as asthma and hypertension. Here, we describe a mechanoresponsive regulatory pathway downstream of tissue stretching that controls contraction of the C. elegans spermatheca, a tubular structure where fertilization occurs. Using live-imaging, we show that ovulation-induced stretching of spermathecal cells leads to recruitment of the RhoGEF RHGF-1 to stress fibers, which activates RHO-1 and myosin II in a positive feedback loop. Through deletion analysis, we identified the PDZ domain of RHGF-1 as responsible for F-actin binding, and genetic epistasis analysis with the RhoGAP spv-1 demonstrated that tension-dependent recruitment of RHGF-1 to F-actin is required for robust spermathecal contractility. Our study illustrates how mechanosensitive regulators of Rho GTPases provide epithelial tubes the ability to tune their contractility in response to internal pressure.

Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Clostridioides Toxins.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.

Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure.

Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.

Ena/VASP proteins in cell edge protrusion, migration and adhesion.

The tightly coordinated, spatiotemporal control of actin filament remodeling provides the basis of fundamental cellular processes, such as cell migration and adhesion. Specific protein assemblies, composed of various actin-binding proteins, are thought to operate in these processes to nucleate and elongate new filaments, arrange them into complex three-dimensional (3D) arrays and recycle them to replenish the actin monomer pool. Actin filament assembly is not only necessary to generate pushing forces against the leading edge membrane or to propel pathogens through the cytoplasm, but also coincides with the generation of stress fibers (SFs) and focal adhesions (FAs) that generate, transmit and sense mechanical tension. The only protein families known to date that directly enhance the elongation of actin filaments are formins and the family of Ena/VASP proteins. Their mechanisms of action, however, in enhancing processive filament elongation are distinct. The aim of this Review is to summarize our current knowledge on the molecular mechanisms of Ena/VASP-mediated actin filament assembly, and to discuss recent insights into the cell biological functions of Ena/VASP proteins in cell edge protrusion, migration and adhesion.

Frameshift mutation S368fs in the gene encoding cytoskeletal ß-actin leads to ACTB-associated syndromic thrombocytopenia by impairing actin dynamics.

Heterozygous dominant mutations in the ubiquitously produced cytoskeletal ß-actin isoform lead to a broad range of human disease phenotypes, which are currently classified as three distinct clinical entities termed Baraitser-Winter-Cerebrofrontofacial syndrome (BWCFF), ACTB-associated pleiotropic malformation syndrome with intellectual disability (ACTB-PMSID), and ACTB-associated syndromic thrombocytopenia (ACTB-AST). The latter two are distinguishable from BWCFF by the presence of milder craniofacial features and less pronounced developmental abnormalities, or the absence of craniofacial features in combination with a characteristic thrombocytopenia with platelet anisotropy. Production and correct function of ß-actin is required for multiple essential processes in all types of cells. Directed cell migration, cytokinesis and morphogenesis are amongst the functions that are supported by ß-actin. Here we report the recombinant production and biochemical characterization of the ACTB-AST mutant p.S368fs, resulting in an altered sequence in the C-terminal region of ß-actin that includes a replacement of the last 8 residues and an elongation of the molecule by 4 residues. The mutation affects a region important for actin polymerization and actin-profilin interaction. Accordingly, we measured markedly reduced rates of nucleation and polymerization during spontaneous actin assembly and lower affinity of p.S368fs for human profilin-1. The reduced affinity is also reflected in the lower propensity of profilin-1 to extend the nucleation phase of p.S368fs. While localized in close proximity to actin-cofilin and actin-myosin interfaces, we determined only minor effects of the mutation on the interaction of mutant filaments with cofilin and myosin family members. However, allosteric effects on sites distant from the mutation manifest themselves in a 7.9 °C reduction in thermal denaturation temperature, a 2-fold increase in the observed IC50 for DNase-I, and changes in nucleotide exchange kinetics. Our results support a disease mechanism involving impaired actin dynamics and function through disruption of actin-profilin interactions and further exacerbated by allosteric perturbations.

VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization.

Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of actin filaments. Members of this protein family have been implicated in a variety of important cellular processes including axon guidance, cell migration and adhesion. However, the specific function of these proteins in macroendocytosis, comprising macropinocytosis and phagocytosis remain rather poorly understood. Here, we used the professional phagocyte Dictyostelium discoideum to address the function and dynamics of its only family member VASP in macroendocytosis. Confocal time-lapse imaging revealed that VASP localized prominently in a circumferential narrow band at the advancing rim of the phagocytic cup followed by its aperture-like convergence upon particle internalization. Loss of VASP resulted in substantial defects in both, macropinocytosis of bulk fluid and phagocytosis of yeast particles. Consistently, VASP-deficiency coincided with diminished speed of the protruding rim and an impaired internalization rate. Most intriguingly, after cup closure, VASP condensed at the distal side of internalized phagosomes and initiated localized de-novo actin assembly to propel the phagosome by an actin-rich comet deeper into the cell, resembling intracellular movement of rocketing Listeria cells. In line with these findings, travelled distance and speed of rocketing phagosomes in VASP-deficient cells were markedly impaired.

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbß3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.

Capping protein is dispensable for polarized actin network growth and actin-based motility.

Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.

Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion.

Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.

Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation.

Efficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin-binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex, an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell-edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.This article has an associated First Person interview with the first author of the paper.

Formins specify membrane patterns generated by propagating actin waves.

Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

Functional integrity of the contractile actin cortex is safeguarded by multiple Diaphanous-related formins.

The contractile actin cortex is a thin layer of filamentous actin, myosin motors, and regulatory proteins beneath the plasma membrane crucial to cytokinesis, morphogenesis, and cell migration. However, the factors regulating actin assembly in this compartment are not well understood. Using the Dictyostelium model system, we show that the three Diaphanous-related formins (DRFs) ForA, ForE, and ForH are regulated by the RhoA-like GTPase RacE and synergize in the assembly of filaments in the actin cortex. Single or double formin-null mutants displayed only moderate defects in cortex function whereas the concurrent elimination of all three formins or of RacE caused massive defects in cortical rigidity and architecture as assessed by aspiration assays and electron microscopy. Consistently, the triple formin and RacE mutants encompassed large peripheral patches devoid of cortical F-actin and exhibited severe defects in cytokinesis and multicellular development. Unexpectedly, many forA- /E-/H- and racE- mutants protruded efficiently, formed multiple exaggerated fronts, and migrated with morphologies reminiscent of rapidly moving fish keratocytes. In 2D-confinement, however, these mutants failed to properly polarize and recruit myosin II to the cell rear essential for migration. Cells arrested in these conditions displayed dramatically amplified flow of cortical actin filaments, as revealed by total internal reflection fluorescence (TIRF) imaging and iterative particle image velocimetry (PIV). Consistently, individual and combined, CRISPR/Cas9-mediated disruption of genes encoding mDia1 and -3 formins in B16-F1 mouse melanoma cells revealed enhanced frequency of cells displaying multiple fronts, again accompanied by defects in cell polarization and migration. These results suggest evolutionarily conserved functions for formin-mediated actin assembly in actin cortex mechanics.

IQGAP-related protein IqgC suppresses Ras signaling during large-scale endocytosis.

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC- and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.

Functional Characterization of Ubiquitin-Like Core Autophagy Protein ATG12 in Dictyostelium discoideum.

Autophagy is a highly conserved intracellular degradative pathway that is crucial for cellular homeostasis. During autophagy, the core autophagy protein ATG12 plays, together with ATG5 and ATG16, an essential role in the expansion of the autophagosomal membrane. In this study we analyzed gene replacement mutants of atg12 in Dictyostelium discoideum AX2 wild-type and ATG16‾ cells. RNAseq analysis revealed a strong enrichment of, firstly, autophagy genes among the up-regulated genes and, secondly, genes implicated in cell motility and phagocytosis among the down-regulated genes in the generated ATG12‾, ATG16‾ and ATG12‾/16‾ cells. The mutant strains showed similar defects in fruiting body formation, autolysosome maturation, and cellular viability, implying that ATG12 and ATG16 act as a functional unit in canonical autophagy. In contrast, ablation of ATG16 or of ATG12 and ATG16 resulted in slightly more severe defects in axenic growth, macropinocytosis, and protein homeostasis than ablation of only ATG12, suggesting that ATG16 fulfils an additional function in these processes. Phagocytosis of yeast, spore viability, and maximal cell density were much more affected in ATG12‾/16‾ cells, indicating that both proteins also have cellular functions independent of each other. In summary, we show that ATG12 and ATG16 fulfil autophagy-independent functions in addition to their role in canonical autophagy.