Molecular Biology of the Cell

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

The SARS-CoV-2 envelope (E) protein is a virulence factor that remodels host endomembranes, but mechanisms remain incompletely understood. We recently demonstrated that E protein interacts with and inhibits the sarco/endoplasmic reticulum Ca2-ATPase (SERCA), disrupting ER calcium homeostasis. Here, we investigated how this perturbation affects autophagy-associated membrane organization. E protein expression induced lipidated LC3 accumulation and enlarged p62-positive structures, consistent with dysregulated autophagic turnover. Although E protein partially colocalized with LC3 and p62, enlarged p62-positive structures were also observed in cells retaining the reticular ER distribution of E protein, indicating that their formation does not require association with E protein or ER reorganization. E protein also increased the association of p62-positive structures with lysosomes without altering lysosome abundance. Pharmacological SERCA activation attenuated E protein-induced remodeling of autophagy-associated structures, demonstrating that SERCA inhibition contributes to these alterations. Together, our findings establish SERCA-dependent ER calcium homeostasis as a host pathway linking E protein expression to remodeling of autophagy-associated membrane compartments, providing a mechanistic framework for how the SARS-CoV-2 E protein promotes ER membrane remodeling associated with coronavirus replication.

Mechanical cues influence cell behavior and fate and are frequently accompanied by changes in nuclear shape; however, how epithelial nuclei accommodate such deformations remains incompletely understood. Here, we investigated the formation and regulation of nuclear wrinkles (NWs), inward folds of the nuclear envelope, in human epithelial cells. Using quantitative confocal imaging in 2.5D spheroid cultures and controlled 2D monolayers, we found that NWs formed frequently in MCF10A cells but rarely in hTERT-RPE1 cells, indicating pronounced cell-type specificity. NW frequency increased with cell density and was tightly associated with coordinated geometric changes consistent with nuclear rounding. Disruption of F-actin organization, but not microtubules, robustly induced NW formation, and acute cell rounding triggered by trypsinization was sufficient to induce widespread wrinkling across multiple cell types. Live-cell imaging revealed that NWs are dynamic and reversible at low cell density but become stabilized under sustained confinement. NW formation occurred without detectable nuclear envelope rupture, DNA damage, or stress-associated histone phosphorylation. Quantitative analysis supports a passive geometric model in which redistribution of excess nuclear surface area accommodates nuclear shape remodeling, allowing epithelial nuclei to buffer mechanical constraints while preserving nuclear integrity.

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

Bipolar spindle assembly is essential for accurate chromosome segregation. KIFC1, a conserved Ran- regulated minus-end-directed kinesin-14 motor, accumulates in the nucleus during interphase and promotes chromatin-mediated spindle assembly during mitosis and meiosis. In human oocytes, reduced KIFC1 levels destabilize meiotic spindles, a defect that can be rescued by increasing KIFC1 expression. However, how KIFC1 expression levels affect mitotic spindle stability during cleavage divisions in vertebrates remains unclear. Here, we show that whereas an approximately 50% reduction in KIFC1 causes no detectable defects in spindle assembly, approximately 10-fold overexpression of KIFC1 induces monopolar spindle formation, leading to chromosome mis-segregation and embryonic lethality in medaka early embryos. KIFC1 overexpression results in ectopic centrosomal localization during interphase, impairing the separation of duplicated centrosomes before mitotic entry. Analyses of KIFC1 mutants demonstrated that these centrosome separation defects require KIFC1s microtubule-binding and motor activities and are further enhanced by deletion of KIFC1s nuclear localization sequences. Together, our findings demonstrate that tight regulation of KIFC1 expression and its nuclear sequestration is essential for the proper separation and positioning of duplicated centrosomes before mitotic entry, thereby ensuring efficient bipolar spindle assembly during the rapid cleavage divisions of vertebrate embryos. HighlightsO_LIKIFC1 accumulates in the nucleus and at the embryonic spindle midplane via the Ran pathway. C_LIO_LIPartial KIFC1 depletion does not impair spindle assembly in medaka early embryos. C_LIO_LIKIFC1 overexpression induces monopolar spindles by preventing centrosome separation. C_LIO_LICentrosome separation defects require KIFC1 microtubule-binding and motor activity. C_LI

Animal microtubule-organizing centers, including the centrosome and the Golgi apparatus, regulate microtubule nucleation and anchoring through the {gamma}-tubulin ring complex ({gamma}-TuRC) and CAMSAP-mediated minus-end stabilization. However, functional redundancy between these pathways has impeded dissection of their contributions to controlling microtubule organization and density. Here, we addressed this problem using combinatorial gene knockouts, protein depletions and Expansion Microscopy. By simultaneously eliminating CAMSAP2 and the {gamma}-TuRC-targeting proteins AKAP450, pericentrin, CDK5RAP2, myomegalin, ninein and AKNA, we generated viable RPE1 cells that lack both Golgi-derived microtubules and {gamma}-TuRC localization within the pericentriolar material and at subdistal appendages. Despite the disruption of these major microtubule-organizing pathways, overall microtubule density was only partially reduced. The remaining microtubules depended on CEP192 and NEDD1, which, together with ch-TOG, can activate {gamma}-TuRC at the centriole wall, in acentriolar cells, and in biochemical reconstitution assays. Our results demonstrate that in the absence of CAMSAP-mediated stabilization, interphase microtubule formation strongly relies on {gamma}-TuRC activation, which occurs through several redundant pathways.

Cellular systems must act robustly to maintain organismal health, including maintaining biophysical properties that allow for appropriate cellular function, and adapting these properties through changes such as those that occur during cell division. However, we still lack tools to measure many of these physical properties with precision in the living cell. For example, the mechanical properties of the nucleoplasm, the fluid-like substance that fills the nucleus, have not been fully characterized. To investigate these properties, we have turned to the fission yeast Schizosaccharomyces pombe (S. pombe), a well-established, genetically tractable model organism that has been used extensively for studying a variety of cell biophysical processes and structures, including the cytoskeleton and cell division. It is an apt system for studying how the nucleus adapts over the course of the cell cycle, since it undergoes closed mitosis, where the nuclear envelope remains intact during cell division. Studying nucleoplasm properties over the course of closed mitosis may help reveal how nuclear volume, shape, surface area expansion, and chromosome segregation are linked and coordinated. To measure nucleoplasm material properties in S. pombe, we have paired Fluorescence Correlation Spectroscopy (FCS) with a photoswitchable fluorophore, enabling fine control over fluorescent intensity inside live cells. We infer material properties from FCS measurements, while the photoswitchable probe enables confocal imaging in conjunction with these measurements, yielding corresponding information about cellular state and dynamics. Interestingly, we find that nucleoplasm material properties do not vary significantly over the cell cycle. Future studies will use this tool to examine how diverse molecular and genetic perturbations alter nucleoplasmic properties, providing insight into how these properties maintain nuclear function and protect genomic integrity over the cell cycle and during development.

The activity-regulated cytoskeleton-associated protein (ARC/ARG3.1) is a key regulator of synaptic plasticity and has both synaptic and nuclear functions. ARC is known to undergo nuclear import and export, yet the dynamic transport behavior of individual ARC particles remains unknown. Using live-cell single-particle tracking, we directly visualize ARC nucleocytoplasmic transport and shuttling in primary hippocampal neurons. Synaptic activation by chemical long-term potentiation (cLTP) treatment increases shuttling behavior and reveals a previously underappreciated organization of ARC within the neuronal cell body cytoplasm, characterized by perinuclear ARC clusters. Disruption of the N-terminal ARC oligomerization motif markedly reduced both perinuclear cluster formation and nucleocytoplasmic shuttling. Together, these findings reveal an activity-dependent relationship between ARC self-assembly, perinuclear organization, and nucleocytoplasmic trafficking, providing a potential mechanism for coordinating the synaptic and nuclear functions of ARC during neuronal plasticity.

Proliferating cells maintain their cytoplasmic density within a narrow range but deviate when entering quiescence or experiencing stress, suggesting active regulation. The mechanisms driving these density adjustments and their impact on cellular function remain unclear. Here, we demonstrate that the conserved cAMP-activated protein kinase A (PKA) is a key regulator of cytoplasmic properties. Inactivation of PKA leads to a drastic increase in cytoplasmic dry mass density and reduced diffusion that depends on the environmental stress response (ESR) transcription factors Msn2/4. This change is mediated by the accumulation of glycogen and trehalose, which have opposing effects on intracellular diffusion. Importantly, the accumulation of these carbohydrates confers stress resistance in distinct ways and independently of their roles as energy sources. Our findings highlight the importance of the biophysical properties of the cytoplasm in stress resistance and the role of glycogen and trehalose in regulating these properties.

Three-dimensional live-cell fluorescence imaging of yeast cells is crucial for studying cell-cycle mechanics and regulation. However, extracting multi-channel phenotypes within dense cell clusters remains an image-processing bottleneck. Standard deep-learning models segment cells but fail to track mother-bud boundaries, mitotic spindle shapes and spindle-localizing proteins. Investigators rely on labour-intensive manual coordinate plotting, introducing observer bias and often exclude clustered cell data due to visual complexity. Here, we present an open-source Fiji pipeline for automated yeast cell image processing and deterministic classification of cell-cycle, spindle and protein dynamics. The workflow utilizes a dual-segmentation architecture via custom Cellpose models to capture the mother-bud cell boundaries. Extracted masks are integrated with multi-channel fluorescence data using a Difference-of-Gaussians framework to resolve SPB coordinates and localized protein kinetics, which a rule-based decision-tree maps to precise mitotic phenotypes. Validation demonstrates a 50-fold acceleration with ~6% deviation from manual analysis. Availability: Zenodo at https://doi.org/10.5281/zenodo.22083016.

Chromosome-bound RCC1 generates Ran-GTP signals to organize functional spindles for faithful chromosome segregation during mitosis and meiosis. RCC1 is the sole guanine nucleotide exchange factor (GEF) for Ran and is essential for spindle assembly during early, but not late, embryonic divisions. However, how RCC1 organizes the specialized embryonic spindle and when its function changes during early embryogenesis remain unclear. Here, using time-resolved RCC1 depletion and depletion-rescue experiments in medaka embryos, we show that RCC1 GEF activity is specifically required before the blastula stage to organize a specialized metaphase spindle mid-plane that ensures faithful chromosome segregation. Mechanistically, RCC1 promotes the accumulation of the canonical Ran effectors HURP and KIFC1/HSET, and unexpectedly, the microtubule motor dynein at the spindle midplane during early embryonic divisions. Intriguingly, a five-fold increase in RCC1 expression phenocopies RCC1 depletion, disrupting spindle-midplane organization and the accumulation of KIFC1 and dynein in a GEF activity-dependent manner. Together, our findings demonstrate that both insufficient and excessive RCC1 GEF activity compromise embryonic spindle assembly, revealing that balanced Ran activation is required to organize the specialized spindle midplane during vertebrate cleavage divisions. HighlightsRCC1 requirement changes with embryonic spindle remodeling before the blastula stage. RCC1 GEF activity is required to organize the specialized embryonic spindle midplane. RCC1 promotes the accumulation of HURP, KIFC1, and dynein at the spindle midplane. Both insufficient and excessive RCC1 GEF activity disrupt the spindle midplane organization.

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

During neurotransmission, synaptic vesicle exocytosis adds membrane and proteins to the cell surface. To sustain further release, this material must be retrieved, via several distinct endocytic modes matched to the level of exocytosis. The GTPase dynamin plays a central role in endocytosis, but it has remained unclear which endocytic modes it supports. In mammals, distinct dynamin gene products with different proline-rich domains (PRDs) are proposed to mediate particular modes of endocytosis; however, the function of each PRD isoform has not been tested in an organism. Drosophila dynamin is encoded by one gene (shibire) that produces long and short PRD isoforms (Shi-L and Shi-S), which differ by a 48 amino acid C-terminal extension. Using isoform-specific knockin and knockdown tools, we found that loss of the more abundant Shi-S isoform disrupted bulk endocytosis and vesicle reformation under high exocytic demand, reduced evoked transmission at moderate levels of activity, and enhanced spontaneous release at rest. These functions did not depend on the PRD extension, as either isoform could rescue these phenotypes when re-expressed. Our results indicate that dynamin contributes to vesicle recycling across multiple endocytic retrieval modes and that PRD specialization is not required for these functions.

The positioning of the mitotic spindle controls the size, content, and position of daughter cells within embryos and tissues. A major spindle positioning mechanism is cortical pulling whereby the membrane-bound complex LGN/NuMA/Dynein captures astral microtubules and pulls centrosomes toward the cell cortex. Cytoplasmic dynein and astral growth tend to counteract cortical pulling and position spindles at the cell center. It remains unclear how these opposing forces cooperate. Here we examine the ascidian embryo, where spindles of two germ line cells rotate toward one another causing divisions which are both mirror symmetric and unequal. We find that this spindle behavior is governed by transient enrichment of LGN and NuMA and enhanced cortical pulling at the shared cell contact. Inhibition of the LGN/NuMA complex disrupts spindle alignment, unequal cleavage, and mirror symmetry. Temporal analysis shows that cortical pulling force initates at anaphase when there is a sharp increase in astral microtubule length. These results point towards two phases of spindle positioning forces, with cytoplasmic pulling and cortical pulling operating sequentially during early and late mitosis.

Previously, we showed that macrophages transfer mitochondria to breast cancer cells, promoting proliferation in acceptor cancer cells. Transferred mitochondria were depolarized and accumulated reactive oxygen species (ROS), and the mitochondrial transfer-induced proliferation was dependent upon ROS signaling (Kidwell et al. 2023). Our unexpected findings supported a model in which transferred mitochondria act as a signal for proliferation in acceptor cancer cells rather than a direct source of increased bioenergetics. It remains unclear whether this unexpected signaling mechanism is unique to macrophages as the donor cell, or whether this mechanism applies to mitochondrial transfer between other cells within the tumor microenvironment. Here, we show that highly metastatic cancer cells transfer mitochondria to weakly metastatic cancer cells. These transferred mitochondria are depolarized, accumulate ROS, and promote ROS-dependent proliferation in acceptor cancer cells. Furthermore, we specifically attribute this proliferative phenotype to the transfer of mitochondria, as when we isolate mitochondria from highly metastatic cells and apply these purified mitochondria directly to weakly metastatic cells in culture, acceptor cancer cells that internalize the purified mitochondria exhibit increased proliferation in a ROS-dependent manner. These findings support mitochondrial transfer within the breast tumor microenvironment as a signaling axis for proliferation, regardless of donor cell identity.

Insulin/IGF signaling (IIS) inhibits the nuclear localization of the DAF-16/FOXO transcription factor to regulate longevity and stress resistance in C. elegans. In the intestine, IIS promotes DAF-16 localization to endosomes and loss of TBC-2, a RAB-5 GAP, results in increased endomembrane localization of DAF-16 at the expense of nuclear localization, decreased DAF-16 target gene expression, longevity and stress resistance. Here we found that TBC-2 differentially regulates the localization of the IIS-regulated transcription factors PQM-1 and HLH-30/TFEB. Our results suggest a broader role for TBC-2 in negatively regulating IIS and that TBC-2 likely functions at an upstream point in the IIS pathway.

The cofactor Fe-protoporphyrin IX cofactor (heme) performs many functions in biology. Animal cells must stabilize their newly generated heme-free (apo)-hemeproteins and deliver mitochondrial heme to them so they can mature to functional form. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) typically accomplishes the heme deliveries, and for many apo-hemeproteins, heat shock protein 90 (Hsp90) drives their heme insertions. We previously observed hemeproteins express poorly in a cell line (COS-7) that does not express soluble guanylyl cyclase (sGC), a heme-binding enzyme that typically functions through its cGMP generation. To understand sGC involvement, we expressed four hemeproteins, Hemoglobin beta (Hb{beta}), Myoglobin (Mb), Indoleamine 2,3-dioxygenase 1 (IDO1), and Tryptophan 2,3-dioxygenase (TDO) in a cell line expressing sGC (HEK293) or in two cell lines (COS-7, DU145) that do not. We assessed hemeprotein expression levels, their abilities to acquire heme, and when relevant if these facets could be rescued by co-expressing individual sGC subunits, including variants with defects in either sGC heme binding, Hsp90 association, heterodimerization, or cGMP production. We found that co-expression of either sGC subunit was essential for three of the four apo-hemeproteins to accumulate in the COS7 and DU145 cells and acquire heme. This did not involve heme binding, heterodimer formation, or cGMP generation by the sGC subunits, and instead depended on a subunits ability to recruit Hsp90 and GAPDH to the apo-hemeproteins via their own Hsp90 binding. Recruiting Hsp90 and GAPDH to apo-hemeprotein clients to ensure they can accumulate and mature to functional form broadens our understanding of sGC and Hsp90 functions in biology.

Cells generate dynamic patterns of Rho GTPase activation to direct the subsequent patterning of Rho GTPase effectors needed to remodel the cell cortex during processes ranging from cell division to cell repair. To understand how such patterns arise, we used live cell imaging, time-resolved Rho GTPase manipulations, and a novel computational tool to study the spatiotemporal dynamics of Rho, Cdc42 and several downstream Cdc42 targets in wounded Xenopus laevis oocytes. We find that the characteristic wound-induced segregation of Rho and Cdc42 activity into concentric zones is followed by polarization of the Cdc42 zone such that Toca-1 progressively concentrates at the back of the Cdc42 zone while Arp2/3, cofilin, cortactin, and the Rho GAP p190RhoGAP progressively concentrate at the front of the Cdc42 zone, where it overlaps the Rho zone. Remarkably, the juxtaposition of Rho activity to Cdc42 is required for the polarization of p190RhoGAP, while p190RhoGAP is responsible for establishing the boundary between the Cdc42 and Rho zones. The results indicate that the characteristic segregation of the Rho and Cdc42 zones, as well as the polarization of the Cdc42 zone, arise from cortical self-organization. Further, these findings reveal a simple mechanism for hierarchical establishment of cortical patterns: recruitment of new proteins to regions of signaling compartment overlap.

The mitochondrial intermembrane space (IMS) is a critical regulatory interface for mitochondrial calcium (mCa2+) flux. Positioned between the outer and inner mitochondrial membranes, the IMS links cytosolic Ca2+ signal to regulated Ca2+ uptake into the matrix. This positioning allows the IMS to influence mCa2+ transport and Ca2+-dependent mitochondrial metabolism. mCa2+ homeostasis is governed mainly by the mitochondrial calcium uniporter complex (mtCU), which mediates mCa2+ uptake, and the Na+/Ca2+ exchanger NCLX, which drives mCa2+ efflux. However, whether IMS regulatory events, particularly proteolytic remodeling by IMS proteases, control this transport machinery remains unclear. Using complementary knockout and overexpression approaches targeting ten IMS proteases (NLN, ATP23, IMMP1L, IMMP2L, YME1L1, OMA1, LACTB2, PARL, and HTRA2), we identified protease-specific remodeling of mtCU components and NCLX abundance. Transcriptomic and proteomic analyses showed that these changes arise largely from protease-specific control of transporter stability rather than transcriptional regulation alone. Proximity-labeling proteomics further revealed spatial associations between IMS proteases and mCa2+ transport components. Functionally, perturbing IMS proteases altered mCa2+ flux and reduced mCa2+ retention capacity, indicating impaired buffering against Ca2+ overload. Together, these findings identify IMS proteases as a proteostatic regulatory network controlling mCa2+ transport and establish a mechanistic link between mitochondrial proteostasis and Ca2+ homeostasis.

Inositol pyrophosphates are conserved signaling molecules synthesized by the bifunctional PPIP5K enzymes, but how their cellular functions diversify across species remains poorly understood. Here, we compared the PPIP5K enzyme Asp1 in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus and in the distantly related fungus Ustilago maydis. All three homologs retained a conserved kinase-phosphatase architecture and catalytic activity. However, whereas Asp1 produced broadly similar effects on actin organization and morphogenesis in S. pombe and U. maydis, its regulatory output was reversed in S. japonicus. In S. pombe and U. maydis, Asp1 positively supported Arp2/3-dependent actin functions, as loss of Asp1 increased sensitivity to the Arp2/3 inhibitor CK666. In contrast, deletion of asp1 in S. japonicus conferred strong CK666 resistance and caused excessive, spatially deregulated actin-patch organization. This opposing cytoskeletal phenotype was mirrored at the level of morphogenesis: Asp1 restricted the yeast-to-hypha transition in S. japonicus, whereas Asp1 was required for pseudohyphal growth in S. pombe and for filamentous development in U. maydis. However, the negative regulatory activity observed in S. japonicus was not an intrinsic property of the SjAsp1 protein. When expressed in S. pombe, SjAsp1 promoted invasive pseudohyphal growth, reproducing the regulatory output of the S. pombe Asp1 morphogenesis pathway rather than that of its native species. Similarly, SjAsp1 supported Arp2/3 functions when expressed in S. pombe. Thus, SjAsp1 adopted the functional behavior imposed by the host cellular environment. S. pombe Asp1 was originally identified as a suppressor of Arp2/3-complex mutant phenotypes, establishing a genetic connection between Asp1 and the actin nucleator. Extending this link, affinity enrichment with inositol pyrophosphates reagents recovered all seven subunits of the S. pombe Arp2/3 complex, providing biochemical support for a potential association between inositol pyrophosphate and Arp2/3. Together, these findings identify S. japonicus as a functional outlier in which a conserved PPIP5K pathway produces an opposing biological output. They further demonstrate that this divergence is determined primarily by species-specific cellular networks rather than by intrinsic differences in the Asp1 protein.