Biology subjects

Swanson, J. A.

Publications and source records attributed to Swanson, J. A..

6 recordsLinked to original sources

bioRxiv2026

PI(3)P Signaling by VPS34 Complex II Orchestrates Macropinocytosis

Macrophage macropinocytosis contributes to wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Here, CRISPR/Cas9 whole-genome screens in murine bone-marrow derived macrophages (BMDM) identified genes regulating unstimulated, PMA-, and CSF1-stimulated uptake of the fluorescent pinocytosis solute tracer, Lucifer yellow. UVRAG and other members of VPS34 complex II (VPS34-II), which catalyze PI(3)P formation from phosphatidylinositol, were identified as positive regulators of macropinocytosis. Targeted gene disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with Uvrag-disrupted BMDM having fewer but larger macropinosomes. In contrast, depletion of ATG14, a unique component of VPS34 complex I, increased solute uptake and the number of macropinosomes. Live-cell imagining of macrophages expressing 2xFYVE-fluorescent protein fusions showed PI(3)P present on the plasma membrane, nascent macropinosomes, and endosomes. The presence of PI(3)P on the plasma membrane prior to macropinocytic cup closure, indicates a novel role on the plasma membrane. Quantitative imaging of fixed cells shows a decrease in concentration of PI(3)P in Uvrag-disrupted BMDMs while increase in concentration of PI(3)P in Atg14-disrupted BMDM compared to WT BMDMs. Treatment with the VPS34 inhibitor SAR-405 acutely decreased macropinocytosis but maintained AKT phosphorylation suggesting class I PI3K activity and PI(3,4,5)P3 production are independent of class III PI3K activity. These results suggest that PI(3)P is a key phosphoinositide governing macropinocytosis at the plasma membrane and that it is primarily formed via direct phosphorylation of PI rather than via the sequential dephosphorylation of PIP3.

cell biology↗

bioRxiv2025

Macropinosomes are a site of HIV-1 entry into primary CD4+ T cells

HIV-1 has been observed to enter target cells at both the plasma membrane and endosomes. However, which pathways mediate its entry into primary CD4+ T cells, the major targets of this virus, remains unclear. Here, we show that HIV-1 can enter primary CD4+ T cells through macropinocytosis, a form of endocytosis. We found that HIV-1 can enter primary CD4+ T cells at both the plasma membrane and internal compartments, while entry into common T cell lines occurred primarily at the plasma membrane. Inhibition of macropinocytosis suppressed HIV-1 internalization into and subsequent fusion with primary CD4+ T cells regardless of the viral coreceptor usage. Microscopic analysis of viral contents exposed to the cytosol confirmed that HIV-1 fusion occurs at the macropinosomal membrane. Finally, the inhibition of macropinocytosis blocked HIV-1 infection of primary CD4+ T cells. Altogether, this study identifies macropinocytosis as one pathway for HIV-1 entry into primary CD4+ T cells. Significance statementHIV-1 entry is an important therapeutic target. However, the exact subcellular location of HIV-1 entry into primary CD4+ T cells, a major in vivo host for HIV-1, remains undetermined. The current study shows that macropinosomes serve as a site for productive HIV-1 entry into the cytoplasm of primary CD4+ T cells. Supporting the role for macropinosomes, inhibition of macropinocytosis prevents HIV-1 internalization into, fusion with, and infection of primary CD4+ T cells. By contrast, HIV-1 infection of a CD4+ T cell line commonly used in HIV-1 research is insensitive to macropinocytosis inhibition. Altogether, this study highlights the primary-T-cell-specific dependence of HIV-1 on macropinocytosis for productive entry and therefore suggests the macropinosome-mediated HIV-1 entry as a potential target for antiviral strategies.

microbiology↗

bioRxiv2025

Light color and nutrients interact to determine freshwater algal community diversity and composition

Lakes experience a wide range of variation in resource availability; variation of essential resources such as light and nutrients can cause changes in algae community structure. Changes in light color and nutrient availability could lead to shifts in algal community diversity and composition, including shifting communities to be dominated by cyanobacteria. Due to eutrophication and brownification, lakes are experiencing increases in nutrient concentrations and shifts from blue towards red in the color of light available to algae for photosynthesis. We investigated whether differences in light color and nutrients affect algal community composition and diversity, and whether differences in light color alter the impact that nutrient levels have on algal communities. We used experimental microcosms with a fully factorial experimental design, crossing four light colors with two nutrient levels. We assessed community composition by enumerating algal taxa via light microscopy. We found that light color and the interaction between light color and nutrient availability led to large differences in community diversity, with blue light leading to the greatest diversity and broad light the lowest. Light color, nutrients, and the interaction between the two were all significant drivers of differences in community composition. Overall, we found that light color and nutrient availability interact to affect algal community diversity, composition, and cyanobacteria density. Consequences of light color are infrequently studied in aquatic ecology, but our results show that light color may need to be considered more broadly. Furthermore, our results suggest that concurrent eutrophication and brownification may yield environmental conditions favorable to cyanobacteria, including taxa that can form harmful algal blooms.

ecology↗

bioRxiv2024

Growing Macrophages Regulate High Rates of Solute Flux by Pinocytosis

In metazoan cells, growth factors stimulate solute ingestion by pinocytosis. To examine the role of pinocytosis in cell growth, this study measured cell proliferation and the attendant rates of solute flux by pinocytosis in murine macrophages in response to the growth factor colony-stimulating factor-1 (CSF1). During CSF1-dependent growth in rich medium, macrophages internalized 72 percent of their cell volume in extracellular fluid every hour. Removal of the essential amino acid leucine from growth medium limited rates of protein synthesis and growth, but increased rates of solute accumulation by macropinocytosis. The amount of protein synthesized during leucine-dependent growth exceeded the capacity of pinocytosis to internalize enough soluble leucine to support growth and proliferation. Fluid-phase solute recycling from lysosomes secreted small molecules from the cells at high rates. Inhibitors of pinocytosis and the mechanistic target-of-rapamycin (mTOR) reduced cell growth and solute recycling, indicating roles for pinocytosis in growth and for nutrient sensing in the regulation of solute flux by pinocytosis. SummaryMurine macrophages growing in response to colony-stimulating factor-1 (CSF1) require pinocytosis. High rates of solute influx and accumulation by pinocytosis are regulated by CSF1 and leucine. Low molecular weight products of protein hydrolysis in lysosomes recycle efficiently from the cells.

cell biology↗

bioRxiv2020

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice lightsheet microscopy

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

cell biology↗

bioRxiv2017

Loss Of PTEN Promotes Formation Of Signaling-Specific Clathrin-Coated Pits

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived signaling-capable CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

cell biology↗