Glioblastoma (GBM) represents the most aggressive primary malignant tumor of the adult central nervous system, with an extremely poor clinical prognosis. As the first-line chemotherapeutic agent for GBM, temozolomide (TMZ) has encountered a critical therapeutic bottleneck due to the development of drug resistance in tumor cells. GBM pathogenesis is jointly driven by multiple factors, including genetics, epigenetics, and the tumor microenvironment. O6-methylguanine-DNA methyltransferase (MGMT) -driven repair of DNA alkylation lesions is the central molecular mechanism underlying TMZ resistance in GBM. Beyond this canonical pathway, the maintenance of stemness in glioma stem cells (GSCs), the bidirectional regulation of autophagy, metabolic reprogramming that reshapes redox homeostasis, and multicellular crosstalk in the tumor microenvironment also contribute to the development of drug resistance. Core molecules such as signal transducer and activator of transcription 3 (STAT3), 5'-AMP-activated protein kinase (AMPK), and mammalian target of rapamycin (mTOR) interact to form an intricate signaling network that modulates drug resistance. This review systematically illustrates the regulatory networks governing GBM pathogenesis and TMZ resistance. It dissects signaling cascades of distinct resistance mechanisms and crosstalk between core signaling axes, and identifies key regulatory molecules and druggable targets for each resistance phenotype. It aims to provide a theoretical basis and novel insights for the development of emerging therapeutic strategies, including targeted therapy, immunomodulation, combination therapy, and nanodrug delivery, thereby promoting the establishment of multi-targeted and individualized precision medicine models and laying a foundation for breaking through the bottleneck of GBM chemoresistance and improving the survival prognosis of patients.
Mitochondria are central hubs of cellular metabolism that harbor their own genome (mtDNA), whose maintenance is essential for both cellular and organismal homeostasis. Unlike nuclear DNA, mtDNA replicates continuously throughout the cell cycle, rendering it particularly sensitive to changes in metabolic state. Emerging evidence indicates that mtDNA homeostasis is not governed solely by dedicated replication factors but is tightly coupled to cellular metabolism. In this review, we discuss how metabolic networks shape mtDNA maintenance through three interconnected layers: mitochondrial nucleotide pools, metabolic control of the replication machinery, and stress-response pathways. This conceptual framework underscores the direct role of metabolic state in governing mtDNA replication, stability, and quality control, with significant implications for mitochondrial disease and therapeutic strategies.
Distinct neuronal subtype specification involves a complex network of transcription factors (TF). Previous studies from our laboratory showed Zinc finger transcription factor of cerebellum (ZIC) 3 to regulate the expression of Tyrosine Hydroxylase (TH), a proxy marker for dopaminergic (DA) neurons, in mouse olfactory bulb (OB) and mid brain (MB) neurons by distinct mechanisms. In absence of ZIC3 consensus binding site in proximal region of mouse TH promoter, ZIC3 interacts with ER81 in OB and regulates TH expression whereas in absence of ER81 in midbrain, ZIC3 regulates TH by enhancing the expression of PITX3. To analyze whether this observation is evolutionarily conserved in humans, different human stem cell model systems were utilized to understand the role of ZIC3 in human DA generation. Differentiation of dental pulp stem cells (DPSCs) with proper cues resulted in the expression of TH. Gain and loss of function demonstrated ZIC3 to be essential for the expression of TH. ZIC3 activates the transcription of TH gene by binding to response element within the region of TH promoter. Addition of SHH, a known morphogen that facilitates dopaminergic differentiation, increased the expression of TH, however, this regulation was suppressed in the absence of ZIC3. Mechanistic insight demonstrated SHH influences ZIC3 expression by GLI protein binding to ZIC3 promoter. Similar role of ZIC3 in TH expression was observed in hiPSCs differentiated to DA like neurons. Conclusively, the present study for the first time demonstrates the undetermined importance of ZIC3 in SHH mediated TH specification in cells of human origin.
Rho GTPase-activating protein 10 (ARHGAP10) is recognized as a tumor suppressor, yet the functional impact of its alternative splicing isoforms on breast cancer metastasis remains unclear. This study aimed to elucidate the role and regulatory mechanism of ARHGAP10 exon 21 skipping in breast cancer progression. Our research results indicate that in metastatic breast cancer cells, the full-length isoform ARHGAP10-L is downregulated, whereas the truncated ARHGAP10-S is upregulated. The RNA-binding protein HNRNPA0 directly binds to intron 21 of ARHGAP10 pre-mRNA, promoting exon-21 skipping and ARHGAP10-S production. Functionally, ARHGAP10-L and ARHGAP10-S exert opposing effects on breast cancer cell malignancy: ARHGAP10-L suppresses migration, invasion, and lung metastasis, whereas ARHGAP10-S promotes these aggressive phenotypes. Moreover, ARHGAP10-S exhibits enhanced binding to CDC42 and is associated with increased AKT phosphorylation. In a nude mouse model, HNRNPA0 drove lung metastasis by upregulating ARHGAP10-S. These findings establish the HNRNPA0-ARHGAP10 splicing axis as a key regulator of breast cancer metastasis, in which ARHGAP10-S promotes progression via the AKT pathway whereas ARHGAP10-L acts as a tumor suppressor, highlighting the therapeutic potential of targeting this splicing event to combat metastasis.
Sushi domain-containing protein 2 (SUSD2) is a transmembrane protein with context-dependent roles in cancer, but its function and mechanism in colorectal cancer (CRC) remain unclear. This study aimed to investigate the role of SUSD2 in CRC progression and its underlying molecular mechanism. Analysis of TCGA dataset revealed that SUSD2 was significantly downregulated in CRC tissues, and low SUSD2 expression correlated with poor patient survival. Functional experiments demonstrated that SUSD2 overexpression suppressed CRC cell proliferation, migration, and invasion, promoted apoptosis in vitro, and inhibited tumor growth in a xenograft mouse model. Mechanistically, SUSD2 activated the NF-κB pathway, leading to p65 nuclear translocation and transcriptional upregulation of NOX4, which induced lethal oxidative stress characterized by increased reactive oxygen species, malondialdehyde, and mitochondrial superoxide. Knockdown of NOX4 rescued SUSD2-mediated phenotypic effects, and pharmacological inhibition of NF-κB abrogated SUSD2-induced NOX4 upregulation, confirming that NOX4 functions downstream of NF-κB in this axis. Collectively, these findings establish SUSD2 as a tumor suppressor in CRC and an independent prognostic biomarker. The SUSD2/NF-κB/NOX4 axis represents a critical regulatory pathway that constrains CRC progression and offers a potential therapeutic vulnerability.
Gliomas remain highly aggressive and treatment-resistant brain tumors, necessitating novel therapeutic strategies. Recent studies have identified cuproptosis, a copper-dependent form of regulated cell death, as a potential vulnerability in cancer. However, the molecular mechanisms regulating cuproptosis in gliomas remain poorly understood. Here, we investigated the role of LAMC1 (Laminin γ1 chain), a laminin subunit implicated in glioma progression, in modulating cuproptosis sensitivity. Multi-dataset bioinformatic analysis revealed LAMC1 as a prognostic biomarker associated with copper homeostasis pathways-high LAMC1 expression correlated with poor survival and effected key cuproptosis-related genes, including FDX1 and LIAS. Functional studies showed that LAMC1 overexpression attenuates copper-induced cytotoxicity, reduces intracellular copper accumulation, and alleviates oxidative stress, whereas LAMC1 knockdown enhances cuproptosis sensitivity, increases ROS production, and disrupts redox balance. Mechanistically, LAMC1 regulates key cuproptosis effectors (FDX1, LIAS, DLAT) and copper transporters (SLC31A1, ATP7B), and modulates the PI3K/AKT signaling pathway under copper stress. The copper chelator TTM reverses the enhanced cuproptosis phenotype induced by LAMC1 knockdown, restoring cell viability and clonogenic capacity while reducing ROS and Cu2+ accumulation and normalizing cuproptosis-related gene expression. These findings establish LAMC1 as a critical regulator of cuproptosis resistance in gliomas, highlighting its potential as a therapeutic target to enhance copper-mediated cytotoxicity in glioma treatment.
The tumor microenvironment plays a critical role in osteosarcoma (OS) progression. However, the mechanisms underlying intercellular communication remain incompletely understood. This study aimed to investigate the role of the APP-CD74 signaling axis in OS and its impact on tumor progression and immune remodeling. Integrated bioinformatics analyses, including bulk RNA-seq, single-cell RNA sequencing, and CellChat, were performed to identify key signaling pathways and cellular sources. In vitro experiments, including wound healing, Transwell invasion, colony formation, ELISA, and Western blot assays, were conducted to assess functional and mechanistic effects. Conditioned medium models and APP neutralization or CD74 knockdown were used to evaluate pathway dependency. A nude mouse xenograft model and immunohistochemistry were employed for in vivo validation. Bioinformatics analyses identified immune-related hub genes and revealed that APP is primarily derived from macrophages and endothelial cells, while CD74 is highly expressed in OS cells. Functional assays indicated that exogenous and microenvironment-derived APP enhanced OS cell migration, invasion, and clonogenicity in a CD74-dependent manner. Mechanistically, APP was associated with the activation of AKT/ERK/NF-κB signaling pathways via CD74. Furthermore, APP-CD74 signaling enhanced the secretion of immunoregulatory cytokines (IL-6, CCL2, TGF-β), driving macrophage polarization toward an M2-like phenotype. In vivo, APP accelerated tumor growth and increased tumor-associated macrophage infiltration, whereas CD74 knockdown markedly attenuated these effects. Collectively, the macrophage/endothelial-derived APP-CD74 axis promotes OS progression by activating tumor-associated signaling and remodeling the immune microenvironment. The APP-CD74 axis therefore warrants further investigation as a candidate therapeutic target.
FBXW7, the substrate recognition component of the SCFFBXW7 E3 ubiquitin ligase complex, functions as a tumor suppressor by regulating cell cycle progression, stem cell maintenance, and cellular differentiation. Loss of FBXW7 contributes to hematopoietic malignancies and promotes monocyte-to-macrophage differentiation. Here, we identify the lineage-determining myeloid transcription factor MAFB as a previously unrecognized substrate of FBXW7. MAFB is a master regulator of monocyte-macrophage lineage commitment, terminal differentiation, and anti-inflammatory macrophage identity. We demonstrate that MAFB protein levels inversely correlate with FBXW7 expression during monocyte-to-macrophage differentiation in both THP-1 cells and primary human peripheral blood mononuclear cells. Consistently, FBXW7 depletion markedly increased MAFB protein abundance, establishing FBXW7 as a key regulator of MAFB stability during myeloid differentiation. Mechanistically, MAFB contains multiple conserved CDC4 phosphodegron motifs overlapping consensus GSK3β phosphorylation sites. FBXW7 directly associated with MAFB and promoted its ubiquitination and proteasomal degradation, whereas an F-box-deficient, ligase-inactive FBXW7 mutant failed to do so. Furthermore, in addition to previously reported GSK3β phosphorylation sites, our mutational analyses identified additional putative phosphodegron residues required for efficient FBXW7 recognition, although their direct phosphorylation by GSK3β remains to be established. Beyond myeloid differentiation, we observed an inverse relationship between FBXW7 and MAFB in multiple myeloma cells, where FBXW7 similarly destabilized MAFB. Collectively, our findings establish the GSK3β-FBXW7-MAFB axis as a conserved post-translational mechanism regulating MAFB stability in myeloid differentiation and multiple myeloma.
Skin flap transplantation is often complicated by ischemia-reperfusion injury, leading to tissue necrosis. Salvianolic acid B (Sa B), a key component of Salvia miltiorrhiza, possesses known antioxidative and anti-apoptotic properties. This study investigated whether Sa B preconditioning could enhance the regenerative capacity of adipose-derived stem cells (ADSCs) before transplantation for improving flap survival. We assessed the effects of Sa B on ADSCs viability, migration, differentiation, and resilience to oxidative stress in vitro under normal and oxygen-glucose deprivation (OGD) conditions. Sa B significantly enhanced ADSC viability, proliferation, and migration, while reducing intracellular ROS accumulation and activating antioxidant responses. A mouse perforator flap model was subsequently established to evaluated the in vivo therapeutic efficacy of Sa B-preconditioned ADSCs. Sa B significantly improved ADSCs viability, proliferation, and migration, while reducing intracellular ROS and activating antioxidant responses. Sa B also promoted ADSCs differentiation towards an endothelial lineage and enhanced angiogenic potential. Mechanistically, Sa B enhanced Nrf2/HO-1-mediated antioxidative defense and promoted PI3K/AKT signaling, resulting in increased anti-apoptotic protein expression and reduced mitochondrial apoptosis. In vivo, transplantation of Sa B-preconditioned ADSCs markedly improved flap survival, tissue regeneration, angiogenesis, and cellular proliferation, accompanied by enhanced antioxidant and anti-apoptotic responses. In conclusion, our findings identify Sa B as a promising pharmacological preconditioning agent that improves the intrinsic regenerative capacity of ADSCs through coordinated antioxidative and anti-apoptotic mechanisms. This strategy provides a potential approach for optimizing ADSCs-based therapy and improving flap survival in reconstructive surgery.
Gefitinib resistance remains a major obstacle to the effective treatment of non-small cell lung cancer (NSCLC), and the underlying molecular mechanisms have not yet been fully elucidated. Lysosomal-associated transmembrane protein 4B (LAPTM4B) has been shown to be involved in cancer progression, but its specific role in gefitinib resistance and the prognosis of NSCLC patients remains unclear. In this study, we conducted functional experiments including CCK-8, Transwell, and tumor sphere formation assays in LAPTM4B-overexpressing or knockdown HCC827 cells and gefitinib-resistant HCC827-R cells. In vivo validation was performed using a NSCLC xenograft model in nude mice. The results showed that LAPTM4B was upregulated in NSCLC, with a further increase in gefitinib-resistant NSCLC samples. Functional experiments demonstrated that knockdown or overexpression of LAPTM4B regulated gefitinib resistance, cell migration, and stemness in HCC827 cells. Furthermore, RPS3 was identified as a key interacting target of LAPTM4B, and LAPTM4B could enhance the stability of RPS3 protein by inhibiting its ubiquitination. In vivo xenograft experiments showed that knockdown of LAPTM4B significantly suppressed tumor growth and tumor stemness, and this inhibitory effect could be reversed by overexpression of RPS3. Collectively, our findings indicate that LAPTM4B promotes gefitinib resistance and NSCLC progression by stabilizing RPS3 protein through inhibiting its ubiquitination, and the LAPTM4B-RPS3 axis may serve as a potential therapeutic target for overcoming gefitinib resistance in NSCLC.
Natural transformation is a key mechanism of bacterial adaptation in which exogenous DNA (eDNA) is taken up, processed into single-stranded DNA (ssDNA), and integrated into the genome. While earlier studies primarily focused on uptake mechanisms, transport proteins, and recombination processes, exonucleases were long regarded as merely nonspecific degradation enzymes in DNA uptake. However, recent studies show that nucleases, partly related to the SOS response, play a key role in processing uptake ssDNA. They affect the imported DNA, thereby promoting efficient recombination. This review highlights the interactions between nucleases and taken-up ssDNA, discusses the functional link between natural competence and the bacterial SOS damage response, and demonstrates that key components of these processes have been conserved in bacteria. This suggests a possible universal principle in which ssDNA-specific nucleases serve as switches between the DNA damage response, competence, and horizontal gene transfer.
This study focused on defining the function and mechanism of non-SMC condensin I complex subunit D2 (NCAPD2) in ovarian cancer progression. NCAPD2 expression in ovarian cancer was analyzed using single-cell data and bioinformatic analysis. With the aim of uncovering the mechanistic role of NCAPD2 in ovarian cancer, we performed gene set enrichment analysis pathway enrichment analysis. To investigate NCAPD2 in ovarian cancer, we quantified its expression in cell lines and clinical specimens using western blot, reverse transcription quantitative polymerase chain reaction, and immunohistochemistry. The functional roles of NCAPD2 were evaluated through cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay staining, wound healing assay and Transwell assay. Western blot analysis was conducted for protein detection. In vivo tumor growth was examined using a mouse xenograft model. Ovarian cancer specimens exhibited elevated NCAPD2 expression. In vitro, NCAPD2 knockdown suppressed proliferation, migration, invasion, and expression of MYC, GRP78, IRE1α, and XBP1s; conversely, its overexpression elicited opposite effects. Furthermore, the suppressive effects of NCAPD2 knockdown on cell proliferation, cell invasion, and expression of GRP78, IRE1α, and XBP1s were partially reversed upon MYC upregulation. In vivo, NCAPD2 knockdown attenuated tumorigenicity. NCAPD2 facilitates ovarian cancer progression, which may be partially involved in MYC-mediated unfolded protein response.
Polyadenylation is a conserved post-transcriptional RNA modification with fundamentally different consequences for RNA fate across biological systems. In bacteria, chloroplasts, and plant mitochondria, adenylation is generally associated with RNA turnover and degradation, whereas its role in metazoan mitochondria remains incompletely understood. In metazoa, polyadenylation is best known for generating complete UAA stop codons in a subset of mitochondrial mRNAs. However, this explanation does not fully account for the evolutionary conservation of the modification, its diverse RNA substrates, or the broad phenotypic consequences of disrupted polyadenylation. In this review, we re-examine RNA adenylation and propose that, in metazoan mitochondria, polyadenylation primarily establishes a permissive 3' end state that governs RNA maturation, stability, translational competence, and decay. This perspective provides a unifying explanation for the diverse functions attributed to mitochondrial polyadenylation.
Sepsis-associated liver injury (SALI) is a serious complication with limited treatment options. While protective autophagy is often suppressed in sepsis, the underlying mechanisms remain unclear. This study investigated whether the SOX4/IGF2BP3/TRIB3 pathway contributes to liver dysfunction by suppressing cytoprotective autophagy. In vitro experiments were performed using LPS-treated mouse primary hepatocytes, with measurements of SOX4, IGF2BP3, and TRIB3 expression by RT-qPCR and Western blot, cell viability by CCK-8, autophagic flux by mRFP-GFP-LC3 probe, and molecular interactions via luciferase reporter, ChIP, RIP, and RNA stability assays. In vivo, a murine sepsis model was established by cecal ligation and puncture. Clinically, mRNA levels of SOX4, IGF2BP3, and TRIB3 were measured in peripheral blood mononuclear cells from sepsis patients and correlated with clinical scores and outcomes. We found that the SOX4/IGF2BP3/TRIB3 axis was upregulated in septic hepatocytes, and its knockdown alleviated LPS-induced injury and impaired autophagic flux. SOX4 transcriptionally activated IGF2BP3, which stabilized TRIB3 mRNA in an m6A-dependent manner. Rescue experiments confirmed that this axis exacerbated LPS-induced hepatocyte injury by suppressing autophagic flux. Inhibition of SOX4 or IGF2BP3 alleviated liver injury and improved survival in septic mice. Clinically, circulating SOX4, IGF2BP3, and TRIB3 levels stratified the presence and severity of SALI and provided high-precision prognostic information. In conclusion, in SALI, SOX4 upregulates IGF2BP3, which stabilizes TRIB3 mRNA in an m6A-dependent manner. Accumulation of TRIB3 protein suppresses cytoprotective autophagy, ultimately leading to hepatocyte death and liver dysfunction.
Mutations in the BRAF (B-raf serine-threonine-protein kinase) gene occur in the majority of melanoma patients and significantly contribute to the progression of the disease. One of the most commonly used methods for treating melanoma is the use of BRAF and MEK (mitogen-activated protein kinase kinase) inhibitors. Unfortunately, resistance to treatment quickly develops in most patients. Therefore, an exhaustive understanding of the molecular basis of this resistance is crucial. We developed two melanoma cell lines resistant to vemurafenib (BRAF inhibitor) and cobimetinib (MEK inhibitor), which we previously characterized as highly invasive. Because cancer cells acquiring resistance may undergo significant metabolic changes, we investigated these processes in the obtained melanoma double-resistant cells. These cells exhibit increased glycolysis and elevated caveolin 1 levels, which contribute to their increased invasiveness. The mitochondria present in the peripheral region of the resistant cells are more functional than those present in the same region of control cells. Resistant cells also form more lipid droplets, but they are smaller than in control cells. Cholesterol ester level is reduced in resistant cells, while free cholesterol level is elevated. Moreover, resistant cells exhibit reduced lipolysis rate and expression of proteins regulating this process.In summary, the obtained results suggest that the purpose of metabolic changes present in melanoma cells resistant to BRAF/MEK inhibitors is mainly to support the significantly increased invasiveness of these cells.
Vascular smooth muscle cells (VSMCs) and human coronary artery endothelial cells (HCAECs) are central mediators of vascular inflammation through NF-κB-dependent transcriptional responses, but the role of the cAMP effector exchange protein directly activated by cAMP 1 (EPAC1) remains unresolved. Here, we combined pharmacological modulation, EPAC1 knockdown, Rap1 activation assays, macrophage-containing vascular tri-culture RNA sequencing, cytokine profiling, and molecular docking to define the contribution of EPAC1 to inflammatory signalling in human vascular cells. In primary VSMCs, forskolin suppressed IL-1β- and TNF-α-induced NF-κB p65 phosphorylation and reduced EPAC1 abundance, confirming a strong anti-inflammatory effect of global cAMP elevation. The non-selective EPAC inhibitor ESI-09 also reduced cytokine-induced p65 Ser536 and Ser529 phosphorylation, whereas the EPAC1-selective inhibitor CE3F4 produced only modest effects on NF-κB signalling despite inhibiting Rap1 activation, confirming functional engagement of EPAC-dependent Rap1 signalling. Conversely, acute activation of EPAC1 using D-007, PWO577 or SY007 increased Rap1-GTP but failed to suppress p65 phosphorylation, and EPAC1 siRNA knockdown did not phenocopy forskolin or ESI-09. In a macrophage-driven tri-culture model, RNA sequencing revealed cell-type- and compound-specific transcriptional responses. ESI-09 produced the largest endothelial DEG burden and a prominent down-regulated VSMC signature, whereas CE3F4 exerted a comparatively modest endothelial effect but increased the VSMC up-regulated DEG pool. Molecular docking supported distinct EPAC ligand-binding modes. Collectively, these findings indicate that cAMP suppresses vascular NF-κB signalling predominantly through EPAC1-independent mechanisms and that ESI-09-associated anti-inflammatory effects should not be interpreted as selective EPAC1 biology alone.
Neutrophilic asthma is defined as an inflammatory disease typified by airway neutrophil infiltration. Tripartite motif-containing protein 31 (TRIM31), an E3 ligase, is implicated in various processes, including inflammation, tumorigenesis, and immune responses. However, the function of TRIM31 in neutrophilic asthma remains to be elucidated. In the study, we identified that TRIM31 expression was markedly upregulated and interleukin 17 (IL-17) pathway had an enrichment in bronchial biopsy tissues from neutrophilic asthma patients through bioinformatics analysis. TRIM31 was also elevated in lung tissues from mice with neutrophilic asthma. Further studies revealed that TRIM31 overexpression inhibited IL-17 mediated signaling cascade. TRIM31 bonded to TRAF6 and the interaction was strengthened in a dose-dependent manner. TRIM31 overexpression reduced the total and M1-linked ubiquitin chains of TRAF6. In the presence of IL-17 A, TRIM31-TRAF6 interaction was enhanced, and TRAF6 ubiquitination further decreased. Whereas TRIM31 knockdown increased TRAF6 ubiquitination and promoted IL-17-mediated signaling activation. In addition, TRIM31 failed to regulate IL-17 signaling in the TRAF6 deficiency cells. Overall, these findings demonstrated that TRIM31 regulated inflammation in neutrophilic asthma through suppression of TRAF6-mediated IL-17 pathway. These results provided new insights into the intricate pathophysiological mechanisms of neutrophilic asthma.
TAK-243, an inhibitor of the ubiquitin-activating enzyme UBA1, impairs the ubiquitination of proteins. We investigated its effects on the intracellular Ca2+ dynamics of human prostate cancer cells DU-145. Live-cell Ca2+ imaging with Fura2 showed that DU-145 cells exhibited spontaneous Ca2+ transients that were largely independent of the activity of the Na+/Ca2+ exchanger but were abolished by extracellular Ca2+ removal, La3+, Gd3+, BTP2 (Orai channel blocker), and thapsigargin (which depletes endoplasmic reticulum -ER- Ca2+ stores). They were also sensitive to U73122 (a phospholipase C inhibitor) and dantrolene (a ryanodine receptor blocker), suggesting the contribution of both store-operated Ca2+ channels and intracellular Ca2+ release pathways in the generation and maintenance of these Ca2+ spikes. At 0.2 μM, TAK-243 augmented the cytosolic concentration of Ca2+ ([Ca2+]i), boosted the Ca2+ spiking activity, whereas at 10 μM it caused a constitutive Ca2+ entry through BTP2-sensitive Ca2+ channels, which further augmented [Ca2+]i but reduced the number of spontaneously active cells. Experiments conducted with the genetically encoded Ca2+ dye CEPIA1er showed that TAK-243 did not empty the ER Ca2+ stores. Moreover, Förster resonance energy transfer (FRET) experiments on HEK-293 cells transfected with Orai1-CFP and STIM1-YFP indicated that it caused the relocalization of STIM1 and its coupling with Orai1. TAK-243 also increased the membrane potential and the Ca2+ buffering capacities of mitochondria. Taken together, treating DU-145 cells with TAK-243 to prevent the ubiquitination of proteins activates the entry of Ca2+ through store-operated Ca2+ channels independently of the ER Ca2+ stores and alters mitochondrial functions.
Microtubule-based organelle transport is essential for organelle positioning within cells and defines the architecture and function of highly polarized cells, like animal neurons and fungal hyphae. Early endosome transport depends on kinesin-3 motors and on cytoplasmic dynein, which binds to this organelle via Hook adaptor proteins. In filamentous fungi, these proteins can propel the indirect transport of additional organelles that hitchhike on early endosomes, like peroxisomes. However, early endosomes carry different cargoes in different fungi, and the contribution of these systems to the subcellular organization of different polarized cells is unclear. Here, we analyzed the function of the kinesin-3 motor KIN2 and of the HOOK1 adaptor in the model fungus Podospora anserina. We found that hyphal growth and morphogenesis require KIN2 and HOOK1, and that early endosome, peroxisome, vacuole, endoplasmic reticulum and mitochondrial motility depends on microtubules. We show that KIN2 and HOOK1 are required for mitochondrial localization at the sites of polarized cell growth, and for the polarized arrangement of the endoplasmic reticulum and vacuoles. Both proteins are required for the bidirectional transport of early endosomes and peroxisomes, but they differently affect their distribution. We found that KIN2 associates with some peroxisomes, and observed a low frequency of peroxisome-early endosome co-transport. Finally, we show that the GTPase RAB5B is required for the distribution and motility of early endosomes but not of peroxisomes, suggesting independent transport systems for these organelles in P. anserina. Our findings reveal a major role for KIN2 and HOOK1 in organelle dynamics during polarized cell growth.
