Journal of Cell Biology

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) coordinate adhesive interactions with intracellular organization during tissue morphogenesis. In the Drosophila follicular epithelium, epithelial maintenance depends on reintegration, a process in which mitotically displaced cells reincorporate into the epithelial monolayer. Previous work identified the IgCAMs Fasciclin 2 (Fas2) and Neuroglian (Nrg) as parallel, partially redundant regulators of reintegration, but the intracellular mechanisms linking adhesion to reintegration remained unclear. Here, we show that Fas2 supports reintegration through two mechanistically distinct modes: a transmembrane mode and a GPI-linked mode. Although both contribute to reintegration, the transmembrane mechanism is more effective and depends on stabilization of a cortical Fas2 pool through intracellular coupling. Using yeast two-hybrid screening, genetics, and fluorescence recovery after photobleaching (FRAP), we identify the scaffold protein Discs large (Dlg1) as a functional intracellular partner of transmembrane Fas2. Partial disruption of Dlg1 preferentially sensitizes epithelia in which the parallel Nrg-dependent reintegration mechanism is compromised, consistent with Dlg1 functioning primarily within the Fas2-dependent reintegration arm. While Dlg1 is not required for Fas2 membrane localization, Dlg1 disruption increases the mobile fraction of transmembrane Fas2, indicating that Dlg1 promotes retention of a stabilized cortical Fas2 pool. Together, these findings support a model in which epithelial reintegration depends on coordinated adhesion-scaffold coupling and reveal mechanistic parallels between epithelial reintegration and IgCAM-dependent processes in the developing nervous system.

Axonal vesicle trafficking is essential for presynaptic assembly, yet the intracellular structures that spatially and temporally regulate vesicle transport during neuronal development remain unclear. Here we identify previously uncharacterized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and both synaptic vesicle precursors (SVPs) and dense-core vesicles (DCVs). Using super-resolution and cryo-electron microscopy, we reveal that vesicle transport undergoes a transient developmental slowdown in the axons of rat hippocampal neurons associated with increased vesicle tethering to the ER. Using proximity biotinylation, we identify the ER protein PRG1 as a developmentally regulated factor enriched at these sites that modulates vesicle mobility. Notably, a disease-associated PRG1 mutant fails to regulate vesicle trafficking. Finally, we demonstrate that PRG1 modulation of vesicle trafficking serves as a developmental checkpoint by delaying synapse formation and preventing premature network activity. Together, our findings establish ER-vesicle contact sites as a previously unrecognized layer of control of axonal trafficking and link intracellular organization to the timing of synapse assembly.

Accurate chromosome segregation requires kinetochores to form robust, load-bearing attachments to dynamic spindle microtubules, mediated primarily by the Ndc80 complex. Two receptors, Dsn1 and Cnn1 (CENP-T), recruit multiple copies of Ndc80c to the kinetochore, but whether they confer functional differences to Ndc80 behavior is unclear. We previously demonstrated that kinetochore components co-purifying with the yeast Dsn1 protein can maintain persistent load-bearing attachments that track with microtubule tip growth and shortening. Using an optical trapping-based assay, we show that Cnn1 purifications also sustain dynamic microtubule attachments under load. Mutation of a conserved region within the disordered N-terminal tail of Cnn1 weakened attachment strength in vitro and caused a growth defect when Dsn1 function was impaired. The Cnn1 mutation reduced Stu2 kinetochore levels without altering other kinetochore proteins. Restoring Stu2, either by direct addition in vitro or by tethering it to Ndc80c in vivo, rescued both attachment strength and cellular viability. These findings reveal a biophysical role for Cnn1 in enabling Stu2-dependent stabilization of kinetochore-microtubule attachments.

Septins are cytoskeletal filaments that associate with the actin and microtubule cytoskeleton, but the mechanisms that govern septin crosstalk and function with these networks are largely unknown. Here, we show that glycogen synthase kinase 3 (GSK3) directly phosphorylates septin-9 (SEPT9), acting as a molecular switch that bidirectionally controls septin distribution between actin and microtubules. We show that GSK3 inhibition redistributes endogenous SEPT9 toward microtubules in multiple cell types. Phosphomimetic mutations at serines 82 and 85 reduce microtubule binding and enhance actin association in cells and in vitro, while phosphonull mutations promote microtubule binding and growth. In primary hippocampal neurons, GSK3{beta} inactivation promotes SEPT9-microtubule association, and phosphomimetic mutations impair asymmetric neurite growth during neuronal polarization. These findings reveal a phosphorylation-dependent mechanism of septin partitioning between actin and microtubules, placing the cytoskeletal functions of septins under the control of GSK3 - a kinase linked to multiple signaling pathways of cell physiology and metabolism. HighlightsO_LIGSK3{beta} phosphorylates SEPT9, and its activity gates septin-cytoskeleton association C_LIO_LIS82/S85 phosphorylation reduce microtubule binding and increase actin localization C_LIO_LIUnphosphorylated SEPT9 binds preferentially to microtubules, promoting their growth C_LIO_LIGSK3{beta} inactivation drives SEPT9 to microtubules to establish neuronal polarity C_LI

Visualizing individual actin filaments in living cells is notoriously difficult. Formins, which track filament plus ends, are powerful probes of actin assembly, yet their behavior in cells is incompletely understood. We dissect the formin mDia1 to reveal how regulatory states control filament assembly. Using TIRF microscopy, we directly compare new regulatory mutants with canonical activity constructs (full-length, FH1-C, FH2-C, and {Delta}DAD). A two amino-acid substitution mutant, mDia1(CA), is constitutively active and drives strong actin nucleation and elongation. In cells, SNAP-mDia1(CA) is enriched in filopodia, where it forms bright, persistent signals at the tips and dim puncta in the shafts. SNAP-mDia1(CA) increases filopodia number but not elongation rate or length, regardless of its use as an overexpression or rescue construct. Although mDia1(CA) is not a universal actin plus-end tracker in this system, its selective enrichment at filopodial plus ends establishes it as a mechanistically distinct probe for interrogating filopodial actin dynamics.

Large secretory vesicles (LSVs) pose a scaling problem for regulated exocytosis. Their micron-scale dimensions greatly increase the vesicular membrane surface area, making productive engagement between the vesicular and target membrane fusion machinery unlikely. Here, we show that vesicular pseudopodia define the fusion sites of LSVs in Drosophila larval salivary glands. Focused ion beam scanning electron microscopy revealed that most LSVs project polarized pseudopodia that interconnect neighboring vesicles and orient toward the apical membrane. Exposed pseudopodia were frequently observed at the apical surface and associated with narrow fusion pores, indicating that fusion occurs at these structures. Three-dimensional correlative light and electron microscopy showed that the I-BAR protein Missing in Metastasis (MIM) selectively localizes to exposed pseudopodia. Proteomic analysis based on a MIM pull-down assay identified exocyst components, including Sec15, which localizes to pseudopodia and persists at fusion sites throughout secretion. Finally, the tetraspanin Tsp42Ee marked complementary apical fusion domains and was required for efficient exocytosis. Our findings support a model in which prepatterned vesicular and apical membrane domains coordinate efficient exocytosis. SummaryRegulated exocytosis of large secretory vesicles is facilitated by vesicular pseudopodia and an apical fusion domain that spatially organizes membrane tethering and fusion during secretion.

The kinetochore and spindle complex (SKA) and NDC80 complexes are essential kinetochore elements that ensure highly accurate chromosome segregation and successful progression through mitosis. The SKA heterodimer complex consists of SKA1, SKA2, and SKA3 subunits, and the NDC80 complex contains NDC80, NUF2, SPC24, and SPC25 subunits. Through live cell fluorescence timelapse imaging assays and expression of RNAi-resistant SKA3 constructs, we rescue SKA complex function in cells lacking endogenous SKA3. These assays reveal a critical span within SKA3s C-terminus required for successful mitotic progression. Structural protein modeling shows that this span encompasses the majority of a roughly 40 amino acid SKA3 C-terminal structural element that promotes interaction with the coiled-coil NDC80 and NUF2 subunits of the NDC80 complex. Thus, although spindle and kinetochore concentration of the SKA complex is mediated in part by the tubulin and tip-tracking capabilities provided by the SKA1 component of the SKA complex, transition from metaphase to anaphase requires the contribution of SKA3s C-terminal structural interface to mediate interaction between the SKA and NDC80 complexes. Significance StatementAccurate chromosome segregation is essential for genomic stability, and its failure underlies developmental defects, and cancer. The kinetochore-microtubule interface, where the SKA and NDC80 complexes converge, is central, yet the of these two complexes is incompletely defined. We identify a critical segment within the C-terminus of SKA3 required for the metaphase-to-anaphase transition. Using live-cell imaging with RNAi-resistant rescue constructs and structural modeling, we demonstrate that this region mediates SKA engagement with the coiled-coil domains of NDC80 and NUF2. Our findings establish that a small segment of SKA3s C-terminus provides an essential physical bridge between the SKA and NDC80 complexes. This work refines understanding kinetochore-microtubule interaction, functionally identifying a discrete contact whose disruption may be relevant to chromosomal instability in disease.

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

Actin-based membrane protrusions such as filopodia, microvilli, and stereocilia support a range of cell functions, from nutrient absorption to mechanosensation. In each case, membrane deformation is supported by a core bundle of actin filaments, organized in a unipolar barbed-end out manner. Although their structures and proteomes are well characterized, mechanisms governing the growth and stability of these protrusions remain less clear. Factors that localize to the distal tips of these structures are of particular interest, as they are well positioned to control actin assembly at filament barbed ends. One such factor, EPS8, localizes to distal tip puncta in multiple protrusion types. While early biochemical studies suggested a role in filament capping, loss of EPS8 in multiple models shortened microvilli and stereocilia, suggesting roles in elongation. More recent studies in differentiating epithelial cells suggested that EPS8 promotes protrusion growth and stability. To clarify EPS8s function in the distal tip compartment, we leveraged acute loss-of-function experiments and titrated gain-of-function approaches in combination with live imaging. Acute sequestration of EPS8 led to rapid depletion of filopodia. Conversely, increasing cellular EPS8 levels elevated EPS8 per distal tip punctum, increased F-actin content within individual filopodia, reduced filopodia elongation rates, increased protrusion lifetimes, and protected filopodia against cytochalasin D-induced collapse. These findings suggest that EPS8 binds filament barbed ends as a leaky capper, slowing monomer addition while stabilizing bundles and preventing collapse. These activities are likely critical for building and maintaining the large arrays of protrusions that are assembled by diverse epithelial cell types.

The spindle checkpoint ensures accurate chromosome segregation by monitoring whether chromosomes, via kinetochores, are properly attached to the spindle. If chromosomes fail to establish bipolar attachment, the checkpoint delays the cell cycle to enable error correction. In C. elegans early embryos, activation of the spindle checkpoint produces a longer mitotic delay in primordial germ cells than somatic cells. We show that the conserved nucleoporin and spindle matrix component, NPP-21/TPR, is required for the stronger spindle checkpoint in germline cells. A checkpoint-proficient NPP-21::GFP transgene localizes to a spindle-like structure during mitosis and is enriched in germline cells, consistent with a cell-fate specific function for this protein. Finally, NPP-21 controls spindle checkpoint strength in germline cells via two, potentially linked, mechanisms: concentrating PCH-2 around mitotic chromosomes and promoting the localization of the checkpoint effector, Mad2, to unattached kinetochores. These experiments demonstrate a developmental role for NPP-21, and the spindle matrix, in controlling spindle checkpoint strength in immortal germline cells in C. elegans.

N-linked glycoprotein processing critically depends on the trimming of -1,2 mannose residues, a key step required both for glycoprotein maturation along the secretory pathway and for targeting defective glycoproteins to endoplasmic reticulum-associated degradation (ERAD). Mammalian cells express seven Class I -1,2 mannosidases, yet their individual roles remain poorly defined, particularly for ManIA, ManIB, and ManIC, which were originally considered Golgi-resident maturation enzymes. Here, we re-evaluated the subcellular localization and functional contributions of these three mannosidases to glycoprotein quality control and maturation. We found that ManIA, ManIB, and ManIC localize predominantly to quality control vesicles (QCVs), previously identified by our group, whereas only ManIC displays a substantial Golgi population. Surprisingly, each enzyme is confined to a different vesicular population. All three enzymes promote ERAD targeting of misfolded model glycoproteins, albeit with different substrate preferences. In addition, they redundantly support the maturation and cell-surface delivery of a model glycoprotein. Most strikingly, in vitro analyses revealed that ManIA, ManIB, and ManIC preferentially trim a properly folded model glycoprotein rather than its denatured form. This is the opposite of the substrate preference that we previously observed for ERManI, EDEM1, and EDEM2. These findings support a model in which ERManI and the EDEMs selectively process misfolded glycoproteins to promote their recognition by the proximal lectin OS-9 and subsequent ERAD. In contrast, ManIA, ManIB and ManIC can slowly process misfolded glycoproteins but act rapidly on properly folded glycoprotein molecules, releasing them from ER-Golgi lectin-mediated retention or retrieval pathways, thereby promoting forward trafficking and maturation in the Golgi.

After endocytosis, transmembrane cargo reaches sorting endosomes where it is partitioned into physically distinct recycling or degradative microdomains. While the J-domain protein RME-8/DNAJC13 is known to maintain these boundaries by actively removing degradative machinery from the recycling microdomain, other factors that contribute to this spatial organization remain poorly defined. Here, we identify the conserved tetraspan protein SCM-1/SCAMP as a key microdomain organizer, discovered through RME-8 proximity-dependent biotinylation screens in C. elegans and human cells. Leveraging the large endosomes of C. elegans coelomocytes, we show that SCM-1 is selectively enriched within the recycling microdomain. In scm-1 mutants, recycling and degradative microdomains still assemble but fail to remain spatially distinct, resulting in inappropriate microdomain overlap. This loss of boundary integrity occurs without increasing the recruitment of sorting machineries, indicating a mechanism distinct from the RME-8-mediated uncoating pathway. scm-1 mutants exhibit significant sorting defects, including misrouting of recycling cargo MIG-14/Wls and v-SNARE SNB-2/VAMP3 to late endosomes and lysosomes. We find that snb-2 mutants themselves missort MIG-14 to late endosomes and lysosomes, suggesting that SNB-2 sorting is key for recycling function. Our data suggest that both microdomains lose efficiency in scm-1 mutants, as cargo missorted into late endosomes and lysosomes is not depleted overall, and degradation of an independent ESCRT-dependent cargo is delayed. We conclude that SCM-1 ensures endosomal sorting fidelity by stabilizing microdomain boundary integrity, a process required for efficient recycling and degradation of transmembrane cargo.

The Arp2/3 complex has long been considered to only assemble branched actin structures in the cell (lamellipodia, endocytic patches, comet tails, and many more). We show for the first time by single-molecule tracking (SMT) that the Arp2/3 complex and SPIN90, which activates Arp2/3 complex to nucleate unbranched filaments, bind to and move in the basal cortex with stress fibers and focal adhesions (FA) that, unlike known sites of Arp2/3 enrichment, employ linear actin bundles. SPIN90 knockout in U2OS cells significantly increases the rate of collective cell migration while decreasing cellular traction (myosin-II and actin speeds) and adhesion (FA size and maturation markers). SPIN90's SH3 domain, similar to its adapter protein Nck1, shows enrichment in FAs, suggesting a possible location for SPIN90-Arp2/3 complex activity. Together, our findings indicate that SPIN90-Arp2/3 nucleated filaments also function in stress fibers where they help define the mechanics of traction and adhesion to regulate cell motility.

Microtubules are polarised polymers that assemble into highly specialised networks in a cell-specific manner. This is controlled in part by microtubule organising centres (MTOCs), which concentrate factors necessary for microtubule nucleation and the organisation of microtubule minus ends. Neurons rely on oppositely polarised microtubule networks, with axons containing mostly plus-end-out microtubules, and dendrites contain many minus-end-out microtubules. How minus-end-out microtubule polarity is established in dendrites remains an important question. Here, we identify a new type of MTOC within the dendrites of Drosophila class I dendritic arborisation neurons, a common model for the neuronal cytoskeleton. We show that membrane swellings distributed intermittently along dendrite shafts, which we term "dendritic varicosities", contain the principal component of the microtubule nucleating complex and repeatedly generate microtubules whose plus ends grow back towards the soma. Varicosities located specifically in distal regions also contain MTOC proteins implicated in minus end anchoring, and this correlates with the accumulation of minus ends specifically in distal varicosities. Depletion of these MTOC proteins leads to major defects in minus end organisation, with microtubule buckles and loops deforming the neuronal membrane. Thus, dendritic varicosities are an important new type of neuronal MTOC that contribute to the generation and organisation of the minus-end-out microtubule network within dendrites.

The microtubule plus-end tracking proteins (+TIPs) CLIP-170/Bik1 and EB/Bim1 form a condensate, the +TIP body, at the plus-end of most microtubules in vivo. Remarkably, however, these +TIP bodies typically impart different dynamics and interaction profiles to distinct microtubules, according to their cellular function. The molecular mechanisms underlying the functional versatility of the +TIP body are unknown. Here, we show that the +TIP Kar9 utilizes repeats of a lysine-aspartate-lysine (KDK)-centered short linear motif (SLiM) to interact with Bik1 on a restricted subset of cytoplasmic microtubules during yeast mitosis. Furthermore, these multivalent Kar9-Bik1 interactions tune the material behavior of the +TIP body to specify proper microtubule function. Indicating that KDK serves as generic Bik1-interaction motif, similar motifs are also present in Kip2, where they mediate Bik1-dependent recruitment of Kip2 to the +TIP body. Together, our study provides insights into how low-affinity Bik1 interactors diversify microtubule function by locally specializing the content and behavior of +TIP bodies.

Right before a cell divides, the centrosome rapidly increases its size and microtubule organizing activity through a process termed centrosome maturation. PCNT, a coiled-coil centrosomal protein, is synthesized and transported to the centrosome simultaneously by dynein. This dynein-mediated co-translational transport of PCNT facilitates centrosome maturation and mitotic spindle formation. How dynein engages and transports PCNT, however, remains unclear. Here we find that PCNT residues 1393-1525 are required for mediating dynein-dynactin interaction. PCNT (1393-1525) also shares similar sequence features with canonical dynein cargo adaptors. PCNT is predicted to interact with dynein heavy chains through similar contact sites used by canonical dynein cargo adaptors. Introducing point mutations at those contact sites abolishes dyneinmediated transport in a peroxisome motility assay. Our results suggest that PCNT contains adaptorlike sequences to bind and activate dynein directly during its transport to the centrosome. Our data also suggest that dynein may engage with some of its cargoes directly without an adaptor.

Centrosomes organize microtubules, coordinate ciliogenesis, and support accurate cell division. At the mature mother centriole, distal and subdistal appendages confer specialized functions in ciliary docking, microtubule organization, and intracellular trafficking. Nuclear distribution element 1 (NDE1) is a centrosome-associated regulator of dynein-dependent processes and ciliogenesis, but its nanoscale organization and contribution to centrosome architecture remain incompletely understood. Here, using expansion microscopy and STED super-resolution imaging, we show that endogenous NDE1 forms a ring-like structure at the subdistal appendages in mouse embryonic fibroblasts and human RPE-1 cells. NDE1 occupies an intermediate radial position between the more centriole-proximal CEP128 layer and the more peripheral ninein layer. Depletion of ODF2 or CEP128 reduces centrosomal NDE1, whereas CEP170 depletion has little effect, placing NDE1 within an ODF2- and CEP128-dependent branch of the subdistal appendage organization network. NDE1 depletion compromises centrosome integrity, reduces the centrosomal enrichment of core centriolar proteins, increases the separation between paired centrioles, and generates ectopic foci containing multiple centriolar markers. Loss of NDE1 also disrupts pericentriolar material organization and impairs the establishment of focused, centrosome-associated microtubule arrays. Furthermore, NDE1 depletion increases LC3B- and p62-positive structures and reduces autophagic flux. Together, our findings establish NDE1 as a subdistal appendage-associated factor that supports centrosome architecture and microtubule-organizing activity. More broadly, they support an emerging view of subdistal appendages as a molecularly layered platform in which distinct but cooperating components connect mother centriole maturation to microtubule organization, ciliary regulation, and intracellular trafficking.

Centrosome amplification is frequently associated with chromosomal instability and tumor progression, but how cells coordinate centriole assembly with the control of centrosome numbers and quality remains poorly understood. TIAM1 is a RAC1 guanine nucleotide exchange factor previously implicated in centrosome-associated signaling and {beta}TrCP-dependent control of PLK4 abundance. Here, we examined how Tiam1 regulates autophagy-lysosome homeostasis in mouse embryonic fibroblasts induced to overexpress PLK4. In contrast to a previous model in which Tiam1 loss promotes productive centriole overduplication, we found, by super-resolution imaging and expansion microscopy, an abnormal distribution of PLK4 on the centrioles centriole-associated structures following TIAM1 depletion, suggesting that TIAM1 may support the organization or maturation of centrioles. TIAM1 depletion also resulted in increased LC3B-positive puncta and enlarged LAMP1-positive compartments, but this was not accompanied by increased LC3B-II accumulation after bafilomycin A1 treatment. These findings suggest that TIAM1 may act at the interface between centriole assembly and endolysosomal/autolysosomal organization, linking TIAM1 to lysosome-associated centrosome quality-control pathways.

Proper cell physiology requires the co-assembly of multiple actin cytoskeletal networks that are tailored for specific functions. To maintain and promote the different functions of these networks, cells decorate them with distinct types of actin binding proteins (ABPs). While various models have been proposed to explain this selective sorting of ABPs, the role of actin disassembly factors is less well understood. Here, we used inducible CRISPR interference and quantitative live-cell imaging to test how disassembly factors control the ABP composition of different networks. We found that knockdown of cofilin (Cof1), a potent and highly conserved disassembly factor, disrupts the size, organization, and ABP composition of actin networks. Specifically, defects in Cof1-mediated disassembly disrupt intracellular transport due to the assembly of overgrown and disordered branched actin networks that are inappropriately decorated by tropomyosin (Tpm1). Contrary to prevailing models of ABP sorting, these networks are co-decorated by Tpm1 and fimbrin (Sac6), and their assembly is independent of formin activity. Instead, our findings support a model wherein failure to maintain the proper architecture of branched actin networks drives mis-localization of network-specific ABPs. Together, this work demonstrates that actin disassembly factors play a critical role in maintaining cytoskeletal structure and function to regulate ABP sorting across distinct networks.

Phagocytosis requires coordinated remodeling of the actin cytoskeleton to generate protrusive and contractile forces that drive target engulfment. Class I myosins Myo1e and Myo1f (Myo1e/f) have been implicated in linking the plasma membrane to the actin network, but their specific roles during Fc-receptor-mediated phagocytosis remain unclear. Using CRISPR-edited RAW 264.7 macrophages lacking Myo1e and Myo1f, we show that double knockout (dKO) cells exhibit markedly reduced uptake of IgG-coated beads, a phenotype that is partially rescued by re-expression of either myosin. Lattice-light-sheet and confocal imaging revealed distinct F-actin architectures corresponding to the various stages of cup progression, including basal podosome-like adhesions, individual phagocytic podosomes (actin teeth) along the rim of the cup, and a contractile phagocytic ring formed by the reorganization of podosomes into a higher-order network. In Myo1e/f- deficient cells, podosome formation was diminished, actin teeth were largely absent, and the phagocytic ring formed prematurely, which was often accompanied by stalled cup progression and repeated engulfment attempts. Myo1e/f localized both to podosomes and to the inner surface of the phagocytic ring, non-muscle myosin II (NM2) localized to the outer surface, and the absence of Myo1e/f correlated with the diffuse distribution of NM2. In addition, Myo1e/f-deficient macrophages exhibited increased trogocytosis of antibody-opsonized HL-60 cells, indicating a shift from whole-target engulfment toward partial target ingestion. These results suggest that Myo1e/f coordinate spatial and temporal transitions between protrusive and contractile actin networks, thereby ensuring efficient phagocytic cup progression. Our findings highlight a dual role for Myo1e/f in adhesion regulation and force balance during macrophage phagocytosis.