Biology subjects

Botelho, R. J.

Publications and source records attributed to Botelho, R. J..

14 recordsLinked to original sources

bioRxiv2026

Mitophagy is required for cell survival upon ultrasound and microbubble therapeutic exposure

Ultrasound-stimulated microbubble (USMB) sonoporation treatment is an emerging strategy for cancer therapy that enhances drug delivery by perforating the plasma membrane. This membrane permeabilization also causes cellular metabolic stress as cells leak metabolites such as ATP and amino acids. Collectively, cells may die in response to excess membrane damage and stress. However, many cells survive the USMB insult by engaging membrane repair and adaptation pathways such as stimulation of AMPK. In this study, we investigated the impact of USMB treatment on mitochondria and their role in post-exposure cellular survival. We found that USMB causes mitochondrial fragmentation and decreases mitochondrial membrane potential. Subsequently, autophagy and Parkin-dependent mitophagy are triggered post-USMB treatment in an AMPK-dependent mechanism. Importantly, Parkin silencing and PINK1 inhibition augment cell death when combined with USMB exposure, suggesting that mitophagy is an important cell survival process in response to USMB. We propose that combining USMB with inhibitors of mitophagy and metabolic stress adaptation is a strong candidate for therapeutic development to treat solid tumours.

cell biology↗

bioRxiv2025

AMPK Repositions Early Endosomes via Gapex-5 to Promote Delivery of Iron to Mitochondria

The regulation of the spatial organization of organelles within cells is critical for coordinating signaling, membrane traffic, and metabolite exchange. Metabolic cues regulate the position and function of lysosomes, yet whether and how metabolic signals may similarly regulate other organelles such as early endosomes (EEs) remains unclear. We find that AMP-activated protein kinase (AMPK), a key regulator of metabolic homeostasis activated in response to nutrient scarcity, triggers movement of EEs to the perinuclear region of cells, leading to enhanced proximity of endosomes to mitochondria and increased delivery of iron to mitochondria. The movement of EEs and increased mitochondrial iron content elicited by AMPK activation requires Gapex-5, a GEF for the early endosome Rab5 previously shown to be an AMPK substrate. These findings reveal a mechanism by which AMPK reprograms endosome positioning to facilitate inter-organelle communication and iron delivery to mitochondria to support metabolic adaptation under conditions of nutrient scarcity.

cell biology↗

bioRxiv2025

Dissipation of lysosome pH impairs formation and collapses existing LPS-induced lysosome tubules in macrophages

While lysosomes are typically globular in morphology, lipopolysaccharide-activated macrophages reorganize lysosomes into an expanded tubular network. Here, we sought to determine if the V-ATPase and the lysosomal pH contribute to LPS-mediated lysosome tubulation in macrophages. We found that inhibition of the V-ATPase prevented lysosome tubulation and collapsed preformed tubules. However, the V-ATPase also controls mTORC1 activity, which itself is needed for tubulation. To distinguish between lysosomal pH and mTORC1, we turned to NH4Cl, which alkalinized lysosomal pH but did not interfere with mTORC1; yet NH4Cl blocked tubulation showing that an acidic lysosomal pH is needed for lysosome remodelling. Moreover, clamping the pH to either acidic or alkaline pH caused tubules to collapse, indicating that the pH gradient promotes tubulation, rather than a specific pH. These effects were not due to altered microtubule organization or impaired lysosome motility, suggesting that motors remained associated with lysosomes. On the other hand, while LPS did not alter the average pH of spherical or tubular lysosomes, growing tubules displayed a more acidic peripheral end relative to the pericentral end. Based on this observation, we propose that a localized pH gradient along the tubule may enable tubulation by modulating factors that catalyse tubule growth.

cell biology↗

bioRxiv2025

PIKfyve governs endoplasmic reticulum-lysosome contacts to modulate endoplasmic reticulum dynamics

Lysosomes clear unwanted cellular material delivered by constant membrane fusion. Membrane fission is thus required to balance lysosome size, number, and composition. PIKfyve is a lipid kinase that converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and promotes lysosome fission since lysosomes coalesce into larger, but fewer organelles in its absence. Here, we reveal a role for PIKfyve in regulating endoplasmic reticulum (ER) dynamics. We show the ER is less reticulated and motile in cells inhibited for PIKfyve. Partly, this arises because lysosomes cluster perinuclearly and are less motile, which appears to arrest ER hitchhiking, a process in which lysosomes pull and form ER tubules. Secondly, the ER morphology is distorted because of hyper-tethering of protrudin, an ER transmembrane protein, to lysosomes via excess PtdIns(3)P and protrudins FYVE domain. Our findings reveal that PIKfyve balances phosphoinositides at ER-lysosome contact sites to govern ER properties and have significant implications for our understanding of PIKfyve function and of diseases linked to its dysfunction. SummaryLong associated with the regulation of the endolysosomal pathway, the authors show that the PIKfyve lipid kinase extends its sphere of influence to modulate the architecture and dynamics of the endoplasmic reticulum by balancing phosphoinositide species at their inter-organellar sites.

cell biology↗

bioRxiv2024

Depletion of endomembrane reservoirs drives phagocytic appetite exhaustion in macrophages

During phagocytosis, a phagocytic cup grows via F-actin remodelling and localized secretion to entrap a particle within a phagosome, which then fuses with endosomes and lysosomes to digest the particle, followed by phagosome resolution. As spatially limited systems, phagocytes have a maximal phagocytic capacity, at which point further uptake must be blunted. However, the processes responsible for phagocytic appetite exhaustion as phagocytes reach their maximal phagocytic capacity are poorly defined. We found that macrophages at their capacity have lower surface levels of Fc{gamma} receptors but overexpression of these receptors did not increase their capacity, suggesting that receptor levels are not limiting. Instead, surface membrane in-folding, membrane tension, and cortical F-actin were all reduced in exhausted macrophages. While this might contribute to appetite suppression, we also found that "free" endosomes and lysosomes were severely depleted in exhausted macrophages. Consequently, focal exocytosis at sites of externally bound particles was blunted. In comparison, macrophages recovered their appetite if phagosome resolution was permitted. We propose that depletion of the endomembrane pools is a major determinant of phagocytic fatigue as macrophages reach their phagocytic capacity. Summary statementMacrophages that reach their maximal phagocytic capacity lose their appetite for further uptake. This appetite exhaustion is driven partly by depletion of endosomes and lysosomes, preventing growth of additional phagocytic cups.

cell biology↗

bioRxiv2024

Control of GSK3beta nuclear localization by amino acid signaling requires GATOR1 but is mTORC1 independent

The availability of certain amino acids regulates cell survival, proliferation, growth, differentiation, and other cellular functions. Sensing of amino acids that converges on the GATOR1 and GATOR2 complexes supports activation of mTORC1 during amino acid replete conditions. Whether amino acid-derived cues regulate additional pathways remains poorly understood. We uncover that amino acid sensing involving GATOR1 and GATOR2 regulates the cellular localization of glycogen synthase kinase 3{beta} (GSK3{beta}). GATOR1 is required to recruit a subset of GSK3{beta} to the lysosome selectively in the presence of amino acids. In addition, while under nutrient replete conditions GSK3{beta} is largely cytosolic, amino acid starvation drives a portion of GSK3{beta} into the nucleus. Acute replenishment of specific amino acids in starved cells triggered nuclear exit of GSK3{beta}. This amino acid-stimulated GSK{beta} nuclear exit required GATOR1 and GATOR2 but was independent of mTORC1 and its activating RagA/B GTPases. This suggests that GATOR1 has a function that diverges from control of mTORC1 to regulate the nucleocytoplasmic shuttling of GSK3{beta}. Furthermore, experimental restriction of GSK3{beta} to the cytoplasm decreased cell survival in amino acid deficient conditions. This suggests that control of GSK3{beta} nuclear localization by GATOR-dependent signals represents a cellular adaptation to metabolic stress that supports cell survival.

cell biology↗

bioRxiv2023

The LCLAT1/LYCAT acyltransferase supports EGF-mediated phosphatidylinositol-3,4,5-trisphosphate and Akt signalling

Receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) stimulate phosphoinositide 3-kinases (PI3Ks) to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3{beta}. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signalling promotes tumour progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signalling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signalling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signalling.

cell biology↗

bioRxiv2022

Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner

The transcription factor TFEB drives expression of lysosomal, autophagic, and immune-responsive genes in response to LPS and phagocytosis. Interestingly, compounds that promote TFEB activity enhance bactericidal activity while intracellular pathogens like Mycobacterium and Salmonella repress TFEB. However, Salmonella enterica sv. Typhimurium (S. Typhimurium) was reported to actively stimulate TFEB, implying a benefit to Salmonella. To better understand the relationship between S. Typhimurium and TFEB, we assessed if S. Typhimurium regulated TFEB in macrophages in a manner dependent on infection conditions. We observed that macrophages that engulfed late-logarithmic grown Salmonella accumulated nuclear TFEB, comparable to macrophages that engulfed E. coli. In contrast, stationary-phase S. Typhimurium infection of macrophages actively delayed TFEB nuclear mobilization. The delay in TFEB nuclear mobilization was not observed in macrophages that engulfed heat-killed stationary-phase Salmonella, or Salmonella lacking functional SPI-1 and SPI-2 type three secretion systems. S. Typhimurium mutated in the master virulence regulator phoP or the secreted effector genes sifA, and sopD also showed TFEB nuclear translocation. Interestingly, while E. coli survived better in tfeb-/- macrophages, S. Typhimurium growth was similar in wild-type and tfeb-/- macrophages. Moreover, Salmonella survival was not readily affected by its growth phase in wild-type or knockout macrophages, though in HeLa cells late-log Salmonella benefitted from the loss of TFEB. Priming macrophages with phagocytosis enhanced the killing of Salmonella in wild-type, but not in tfeb-/- macrophages. Collectively, S. Typhimurium orchestrate TFEB in a manner dependent on infection conditions, while disturbing this context-dependent control of TFEB may be detrimental to Salmonella survival. ImportanceActivation of the host transcription factor TFEB helps mammalian cells adapt to stresses such as starvation and infection by upregulating lysosome, autophagy, and immuno-protective gene expression. Thus, TFEB is generally thought to protect host cells. However, it may also be that pathogenic bacteria like Salmonella orchestrate TFEB in a spatio-temporal manner to harness its functions to grow intracellularly. Indeed, the relationship between Salmonella and TFEB is controversial since some studies showed that Salmonella actively promotes TFEB, while others have observed that Salmonella degrades TFEB and that compounds that promote TFEB restrict bacterial growth. Our work provides a path to resolve these apparent discordant observations since we showed that stationary-grown Salmonella actively delays TFEB after infection, while late-log Salmonella is permissive of TFEB activation. Nevertheless, the exact function of this manipulation remains unclear, but conditions that erase the conditional control of TFEB by Salmonella may be detrimental to the microbe.

cell biology↗

bioRxiv2022

AMP-activated protein kinase (AMPK) is required for recovery from metabolic stress induced by ultrasound microbubble treatment

Ultrasound and microbubbles (USMB) is a promising strategy for cancer therapy. USMB can induce a variety of effects on cells including transient formation of plasma membrane pores (sonoporation) and enhanced endocytosis, which enhance drug delivery, and can also lead to enhanced cell death. However, the outcomes of USMB on cell physiology are heterogeneous, in that USMB elicits cell death in a proportion of cells while exerting minimal effects on others. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. The molecular mechanisms of adaptation to USMB-induced stress remain poorly understood, thus potentially hindering broad therapeutic applications of USMB. Herein, we used several triple negative breast cancer cells to study the effect of USMB-induced metabolite stress and the role of AMPK as a response to this stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates. USMB treatment acutely reduces ATP levels and stimulates AMP-activated protein kinase (AMPK) phosphorylation and activation. Further, AMPK is required to restore ATP levels in cells that survived the initial insult and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the anti-neoplastic efficacy of USMB treatment.

cell biology↗

bioRxiv2021

PIKfyve inhibition reveals a novel role for Inpp4b in the regulation of PtdIns(3)P and lysosome dynamics

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that co-ordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the action of diverse PtdIns kinases and phosphatases. Specific roles for PtdIns in lysosomal function and dynamics are currently unclear and require further investigation. PIKfyve, a lipid kinase which synthesizes PtdIns(3,5)P2 from PtdIns(3)P, controls lysosome "fusion-fission" cycles dynamics, autophagosome turnover and endocytic cargo delivery. We have recently characterized a role for INPP4B, a PtdIns phosphatase which hydrolyses PtdIns(3,4)P2 to form PtdIns(3)P, in the regulation of lysosomal biogenesis and function. To gain a better understanding of PtdIns homeostasis on lysosomes, we investigated the consequence of disrupting PIKfyve in Inpp4b-deficient mouse embryonic fibroblasts. Surprisingly, simultaneous inhibition of Inpp4b and PIKfyve functions impair lysosome "fission" dynamics and thereby exacerbate lysosome enlargement, inhibit autophagic flux. Further examination into the underlying processes that may explain exaggerated lysosome enlargement revealed elevated levels of lysosome associated PtdIns(3)P as contributing factors that control lysosome morphology in cells where Inpp4b and PIKfyve are disrupted. Overall, our study suggests that lysosomal functions are regulated by Inpp4b, through a paradoxical role in suppressing the induction of PtdIns(3)P production. SUMMARY STATEMENTIn this study we identify a novel crosstalk between Inpp4b and PIKfyve. This crosstalk regulates lysosome membrane phosphoinositide composition and lysosomal homeostasis. Our data demonstrate that Inpp4b restricts VPS34-dependent induction of PtdIns(3)P levels, which is activated upon apilimod-mediated PIKfyve inhibition. Through this mechanism, Inpp4b contributes to the regulation of lysosomal membrane dynamics and homeostasis.

cell biology↗

bioRxiv2021

INPP4B promotes leukemia by restricting leukemic stem cell differentiation through regulation of lysosomal functions

Signaling pathways that control vital features of leukemic stem cells including multipotency, self-renewal, clonal expansion and quiescence remain unclear. Emerging studies illustrate critical roles for lysosomes in hematopoietic and leukemic stem cell fate. By investigating consequences of INPP4B alterations in AML, we have discovered its role in driving leukemic stemness. We observed that INPP4B is highly expressed leukemic stem cell populations and Inpp4b-deficeint leukemias demonstrate increased disease latency, reduced leukemia initiating potential which is associated with a differentiated leukemic phenotype. Molecular analyses show that Inpp4b-deficient leukemias have compromised lysosomal gene expression, lysosomal content, and lysosomal activity. Our discovery of a novel pathway linking INPP4B, lysosomal biogenesis and leukemic stemness, provides a mechanism to explain the association of high INPP4B expression with poor AML prognosis, and highlights novel patient stratification strategies and LSC-specific leukemic therapies. Key PointsOur findings highlight a novel pathway linking INPP4B, lysosomal function and leukemic stemness that explains the prognostic role of INPP4B in AML. Our data reveal the utility of INPP4B as a biomarker of aggressive AML and provide a rationale to explore INPP4B and its associated function in lysosome biology as novel strategies to target LSC and AML

cancer biology↗

bioRxiv2020

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as phagosome resolution. Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintain the degradative power of macrophages to handle multiple waves of phagocytosis. SummaryPhagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes fragment to reform lysosomes in a clathrin-dependent manner. This process helps maintain the degradative capacity of phagocytes for subsequent rounds of phagocytosis.

cell biology↗

bioRxiv2019

Reactive oxygen species rescue lysosome coalescence during PIKfyve inhibition

Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and accelerated lysosome fragmentation during re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, glutathione, and thioredoxin that produce superoxide, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.

cell biology↗

bioRxiv2018

Lysosome enlargement during inhibition of the lipid kinase PIKfyve proceeds through lysosome coalescence

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux, and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects including embryonic lethality, neurodegeneration and overt inflammation. While being the most dramatic phenotype, the reasons for lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.\n\nSummary statementPIKfyve inhibition causes lysosomes to coalesce, resulting in fewer, enlarged lysosomes. We also show that TFEB-mediated lysosome biosynthesis does not contribute to swelling.

cell biology↗