Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown mechanisms modulating TRAIL activity in patients. We hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. RNAseq analysis of MDA-MB-231 cells along with validation in additional cell lines demonstrated that TRAIL induced cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, TRAIL dependent induction of the cytokines was predominantly mediated by death receptor 5, caspase-8 and the non-canonical NFKB2 pathway. These cytokines produced by TRAIL-treated TNBC cells enhanced chemotaxis of normal human donor isolated neutrophils. Using TNBC xenograft models, TRAIL induced activation of NFkB2 pathway, cytokine production and increased neutrophil recruitment into the tumors. Moreover, preincubation of neutrophils in supernatants from TRAIL-treated TNBC cells significantly impaired neutrophil function as measured by reduced respiratory burst and cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies showed that these neutrophils suppress T cell proliferation and augment Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and neutrophil-mediated immune suppression.

Glycolipid-lectin-driven endocytosis controls the formation of clathrin-independent carriers and the internalization of various cargos such as beta 1 integrin. Whether this process is regulated in a dynamic manner remained unexplored. Here we demonstrate that, within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell-surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process-mediated by the Na+/H+ antiporter NHE1 as well as the neuraminidases Neu1 and Neu3-requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. De-sialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan make-up is reset to regulate EGF-dependent invasive-cell migration. Further evidence is provided for a role of neuraminidases and galectin-3 in acidification-dependent bone resorption. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 347.

Abstract Genomic instability is a hallmark of cancer. Unlike normal diploid cells, cancer cells exhibit abnormal centrosome numbers and chromosomal instability. Mitosis with supernumerary centrosomes yields progeny with aberrant chromosome segregation and aneuploidy. While most progeny cells with abnormal chromosomes are eliminated through apoptosis, some have distinct fates with a growth advantage. These cancer cells can acquire resistance to antineoplastic agents. This confers an aggressive tumor biology and unfavorable clinical outcomes. Cyclin-dependent kinase 2 (CDK2) regulates cell cycle progression and the centrosome cycle. We previously reported that CDK2 inhibition prevents supernumerary centrosome clustering, causing multipolar mitosis and anaphase catastrophe in lung and other aneuploid cancers. This was associated with residual transplanted tumors in mice. To elucidate cell fates of progeny with multipolar mitosis after CDK2/9 inhibition with CYC065 (0.2 µM) treatment, a panel of murine and human lung cancer cells underwent time-lapse fluorescent microscopy. This elucidated multipolar mitotic events within the progeny. Intriguingly, distinct outcomes occurred after mitosis including formation of multipolar and multinucleated cells. Cell death of progeny is the predominant fate, but some progeny survive despite continuous CDK2 inhibition. Surprisingly, some of these cells fuse together. Those multinucleated cells can undergo multiple cell cycles without successful cytokinesis. To confirm these effects were through CDK2 inhibition, the selective CDK2 inhibitor Tagtociclib (PF-07104091, at the 2 µM dosage) was used. This treatment statistically-significantly increased multipolar lung cancer cells and increased apoptotic death during time-lapse fluorescent microscopy. Findings were independently validated by CDK2 shRNA knockdown. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent staining revealed the ultrastructure of multinucleated cells. Insights into tumor biology came from intravital imaging of transplanted lung cancer cells in mice. Multipolar and multinuclear cells followed CDK2 antagonism. Incucyte® Live-Cell Analysis System and Artificial Intelligence-based imaging determined distinct growth responses in aneuploid as compared to non-aneuploid lung cancer cells after CDK2 inhibitor treatments. In summary, these findings are translationally relevant. CDK2 inhibition of aneuploid lung cancers yields distinct cell fates. This is linked to resistance to CDK2 antagonism and to formation of residual in vivo tumors. Citation Format: Liliya Tyutyunyk, Zibo Chen, Xiuxia Liu, Yeap Ng, Aayush Bhatawadekar, Kedar Narayan, Roberto Weigert, Xi Liu, Ethan Dmitrovsky. Elucidation of the fates of CDK2 inhibited aneuploid and residual lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2117.

PAX3/7 fusion-negative rhabdomyosarcoma (FN-RMS) is a childhood mesodermal lineage malignancy with a poor prognosis for metastatic or relapsed cases. Limited understanding of advanced FN-RMS is partially attributed to the absence of sequential invasion and dissemination events and the challenge in studying cell behavior, using, for example, non-invasive intravital microscopy (IVM), in currently used xenograft models. Here, we developed an orthotopic tongue xenograft model of FN-RMS to study cell behavior and the molecular basis of invasion and metastasis using IVM. FN-RMS cells are retained in the tongue and invade locally into muscle mysial spaces and vascular lumen, with evidence of hematogenous dissemination to the lungs and lymphatic dissemination to lymph nodes. Using IVM of tongue xenografts reveals shifts in cellular phenotype, migration to blood and lymphatic vessels, and lymphatic intravasation. Insight from this model into tumor invasion and metastasis at the tissue, cellular, and subcellular level can guide new therapeutic avenues for advanced FN-RMS.

Dear Editor, Pompe disease (acid alpha-glucosidase [GAA] deficiency) is a severe multisystem lysosomal glycogen storage disorder that is primarily marked by progressive deterioration of muscle tissues.1 The limited efficacy of enzyme replacement therapy (ERT) – the only available treatment option – spearheaded efforts to develop gene therapy approaches and new drugs with improved muscle targeting for ERT.1 Over the years, our studies using a mouse model (knockout [KO]) provided a large body of evidence indicating that: 1) the dysfunction of glycogen-laden lysosomes leads to a major secondary abnormality in the diseased muscle – impaired autophagy and massive autophagic buildup; 2) the buildup resolution upon treatments occurs only when glycogen levels and lysosomal pool return to normal; and 3) the elimination of autophagic buildup is a reliable indicator of therapeutic efficacy.2-4 We have recently reported that a newly developed recombinant adeno-associated virus (rAAV)-based systemic gene therapy resulted in a fast and long-lasting reversal of pathology in multiple tissues of KO mice, including the limb muscle (gastrocnemius).2 Preclinical testing of new therapies is a lengthy undertaking that requires a large number of animals and involves an array of techniques to analyze samples ex vivo. High-resolution intravital microscopy (IVM), a powerful technique with a wide range of research applications, offers the possibility to visualize and quantify the effectiveness of new treatments in live animals within the natural tissue context. In this study, we revisited the same gene therapy approach2 and applied IVM to peer inside living muscle cells in a reporter KO model expressing green fluorescent protein (GFP) fused to autophagosomal marker LC3. The IVM imaging of gastrocnemius muscle was performed seven weeks after a single intravenous administration of an AAV9 vector (2.5 × 1013 vg/kg) expressing human GAA transgene. The animals were 5–7 months of age at the start of therapy. (The mouse strain, referred to as GFP-LC3:KO, and the vector are described in Supporting Information.) As expected, autophagic accumulation spanning along the longitudinal fibre axis can be seen in virtually all myofibers (94.6 ± 5.8%; n = 122 from three animals) of untreated GFP-LC3:KO (Figure 1A–C, GFP-LC3 middle panels). Consistent with our previous data,2 gene therapy reversed the pathology, as indicated by near complete elimination of autophagic buildup – 93.1 ±6.3 % fibres (n = 112 from four animals) were buildup-free (Figure 1A–C, GFP-LC3 bottom panels; Videos S1–S3). However, unlike in the previous study, the reversal can be directly observed in live GFP-LC3:KO without laborious biochemical and molecular biology techniques and with a much-reduced number of animals. Furthermore, we took advantage of the fact that the GFP signal can be collected together with the NAD(P)H fluorescence signal; endogenous NAD(P)H can be excited by two-photon (2P) microscopy and used to measure mitochondrial function and metabolic activity in live animals at subcellular resolution.5-7 The observed NAD(P)H fluorescence intensity in the buildup areas in the muscle of GFP-LC3:KO appeared weaker than in the neighbouring buildup-free regions (Figure 1C). Quantitative measurements (Supporting Information) confirmed visual observations – NAD(P)H levels were significantly reduced in the buildup areas (Figure 1D), in agreement with the disruption of mitochondrial morphology/function previously reported by us and others.8, 9 Next, we explored the possibility of evaluating therapeutic efficacy by using non-invasive imaging of the tongue muscle as a substitute for skeletal muscle (tongue involvement in Pompe patients is described in Supporting Information). This would allow for extended follow-up to monitor the disease progression and the effect of therapies. In preliminary experiments, we used a conventional ex vivo method – confocal microscopy of fixed single fibres isolated from the tongue of wild-type (WT) and KO mice. Immunostaining with LAMP1 (lysosomal marker) and LC3 showed small dot-like structures in WT fibres (Figure 2A). In contrast, LAMP1-positive enlarged lysosomes, and blurry areas (arrows) corresponding to the buildup, were detected in the tongue muscle of KO mice (Figure 2B). LAMP1/LC3 immunostaining revealed enlarged lysosomes and lengthy areas of autophagic buildup of different shapes in KO (Figure 2C). Thus, the pathogenic mechanism in the diseased tongue muscle, much like in skeletal muscle, involves both lysosomal and autophagic pathologies. IVM imaging of the tongue was performed as described in Supporting Information. The muscle layer, which is located ∼100 μm below the surface of the tongue beneath the epithelium and a layer of collagen I (Figure 3A), is well within the imaging range of 2P microscopy.10 First, we imaged the two-thirds of the ventral side of the tongue at low magnification using the tiling mode to collect a large field of view (5145 × 7055 μm), which revealed bright GFP-LC3 streaks throughout the tissue in GFP-LC3:KO (Figure 3B; expanded images in Figure S1). Next, we acquired 42 μm z-stacks below the collagen layer at higher magnification to visualize the myofibers (Figure 3C,D; Videos S4 and S5). Patches of GFP-LC3, compatible in shape and distribution with autophagic buildup appeared throughout the fibres, but only in GFP-LC3:KO. In control GFP-LC3:WT mice (5.5-month-old), we observed scattered GFP-LC3 puncta (Figure 3D, arrows), consistent with basal levels of autophagy. The NAD(P)H levels were significantly reduced in the buildup areas (Figure 3E). Analysis of age-dependent changes revealed a slight increase in the number of fibres with autophagic buildup in 7-month-old compared to 3.5-month-old mice followed by a significant increase in 11-month-old animals, in which ∼80% of fibres were affected (Figure 3F). As in the limb muscle, the buildup resolution was observed in the tongue muscle of GFP-LC3:KO mice seven weeks after the start of gene therapy (Figure 4 and Figure S2). The vast majority of fibres (97.8 ± 1.0%; n = 220 from four animals) were buildup-free. Finally, we treated 3.5-month-old GFP-LC3:KO (n = 2) and imaged the tongue 15 weeks after dosing (Figure S2; Video 6); once again, 98.9 ± 2.2% myofibers (n = 81 from two animals) were buildup-free, indicating that the therapy reversed the autophagy defect and halted its progression. Thus, the therapy was equally successful in rescuing pathology in both limb and tongue muscles. In summary, this is the first report of IVM application to visualize muscle damage in Pompe disease. The reporter model enables monitoring of the disease progression and response to therapeutic interventions by non-invasive imaging of the tongue muscle. Naresh K. Meena, Yeap Ng and Davide Randazzo performed experiments, analyzed, and interpreted the data; Roberto Weigert supervised the research and contributed to writing the manuscript; Rosa Puertollano provided funding and supervised the research; Nina Raben designed the study, performed experiments, analyzed, and interpreted the data, and wrote the paper. We are grateful to our colleagues at Amicus Therapeutics for providing the vector for gene therapy. This research was supported by the Intramural Research Program of the NHLBI of the National Institutes of Health (ZIA HL000140). Naresh K. Meena was supported in part by a CRADA between NIH and Amicus Therapeutics. The authors declare no conflict of interest. All experiments were performed in accordance with the guidelines provided by the National Cancer Institute and the National Heart, Lung and Blood Institute (National Institutes of Health, Bethesda, MD, USA) Animal Care and Use Committies (ACUC) and were compliant with all relevant ethical regulations regarding animal research. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

ASAP1 is a multidomain ADP-ribosylation factor (Arf) GTPase-activating protein (GAP) involved in the regulation of the actin cytoskeleton, focal adhesions dynamics and receptor tyrosine kinases trafficking. ASAP1 expression levels correlate with progression of solid tumors (e.g. breast, colorectal, pancreatic, ovarian, gastric, and prostate cancer). The first evidence for a role of ASAP1 in cancer progression was the discovery that the amplification of ASAP1 gene on chromosome 8 correlates with poor prognosis in uveal melanoma. A similar amplification of chromosome 8 has been reported in ~72% of fusion negative rhabdomyosarcoma (FN-RMS) tumors, suggesting a potential role for ASAP1 signaling in the progression of FN-RMS. To determine the function of ASAP1 in tumor growth, invasion and metastasis, we used a tongue orthotopic xenograft of FN-RMS which recapitulates physiologically relevant tumor progression and metastasis to lymph nodes and lungs, two of the common metastatic sites in patients. Using intravital microscopy, we observed that injected FN-RMS cells formed elongated structures and exhibited dynamic branching, which might represent local invasion. H&E stained of fixed tissue revealed invasion into the tongue and local metastasis. Metastases to the regional lymph nodes, lower mandible and lungs were also observed. Using this system, we are now able to determine effect of knocking down and knocking out ASAP1 on the progression of the xenografts, including invasion in real time and metastasis. Citation Format: Sarah M. Hammoudeh, Yeap Ng, Mukesh P. Yadav, Roberto Weigert, Paul A. Randazzo. The use of a novel orthotopic xenograft model of fusion negative rhabdomyosarcoma to study the effect of ASAP1 on tumor progression and metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3589.

Abstract ASAP1 is a multidomain ADP-ribosylation factor (Arf) GTPase-activating protein (GAP) involved in the regulation of the actin cytoskeleton, focal adhesions dynamics and receptor tyrosine kinases trafficking. ASAP1 expression levels correlate with progression of solid tumors (e.g. breast, colorectal, pancreatic, ovarian, gastric, and prostate cancer). The first evidence for a role of ASAP1 in cancer progression was the discovery that the amplification of ASAP1 gene on chromosome 8 correlates with poor prognosis in uveal melanoma. A similar amplification of chromosome 8 has been reported in ~72% of fusion negative rhabdomyosarcoma (FN-RMS) tumors, suggesting a potential role for ASAP1 signaling in the progression of FN-RMS. To determine the function of ASAP1 in tumor growth, invasion and metastasis, we used a tongue orthotopic xenograft of FN-RMS which recapitulates physiologically relevant tumor progression and metastasis to lymph nodes and lungs, two of the common metastatic sites in patients. Using intravital microscopy, we observed that injected FN-RMS cells formed elongated structures and exhibited dynamic branching, which might represent local invasion. H&E stained of fixed tissue revealed invasion into the tongue and local metastasis. Metastases to the regional lymph nodes, lower mandible and lungs were also observed. Using this system, we are now able to determine effect of knocking down and knocking out ASAP1 on the progression of the xenografts, including invasion in real time and metastasis. Citation Format: Sarah M. Hammoudeh, Yeap Ng, Mukesh P. Yadav, Roberto Weigert, Paul A. Randazzo. The use of a novel orthotopic xenograft model of fusion negative rhabdomyosarcoma to study the effect of ASAP1 on tumor progression and metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3589.

Summary It is commonly assumed that the glycan makeup of glycoproteins that reach the cell surface is final and static. Here, we challenge this notion by the discovery of a molecular switch that induces acute and reversible changes of glycans on the plasma membrane. We demonstrate that within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process, mediated by the Na + /H + antiporter NHE-1 and the neuraminidases Neu1/3, requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. Desialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan makeup is reset, and their function is repurposed to regulate EGF-dependent invasive cell migration. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

Localization of RNAs at protrusive regions of cells is important for single-cell migration on two-dimensional surfaces. Protrusion-enriched RNAs encode factors linked to cancer progression, such as the RAB13 GTPase and the NET1 guanine nucleotide exchange factor, and are regulated by the tumor-suppressor protein APC. However, tumor cells in vivo often do not move as single cells but rather utilize collective modes of invasion and dissemination. Here, we developed an inducible system of three-dimensional (3D) collective invasion to study the behavior and importance of protrusion-enriched RNAs. We find that, strikingly, both the RAB13 and NET1 RNAs are enriched specifically at the invasive front of leader cells in invasive cell strands. This localization requires microtubules and coincides with sites of high laminin concentration. Indeed, laminin association and integrin engagement are required for RNA accumulation at the invasive front. Importantly, perturbing RNA accumulation reduces collective 3D invasion. Examination of in vivo tumors reveals a similar localization of the RAB13 and NET1 RNAs at potential invasive sites, suggesting that this mechanism could provide a targeting opportunity for interfering with collective cancer cell invasion.

Actomyosin networks, the cell's major force production machineries, remodel cellular membranes during myriad dynamic processes(1,2) by assembling into various architectures with distinct force generation properties(3,4). While linear and branched actomyosin architectures are well characterized in cell-culture and cell-free systems(3), it is not known how actin and myosin networks form and function to remodel membranes in complex three-dimensional mammalian tissues. Here, we use four-dimensional spinning-disc confocal microscopy with image deconvolution to acquire macromolecular-scale detail of dynamic actomyosin networks in exocrine glands of live mice. We address how actin and myosin organize around large membrane-bound secretory vesicles and generate the forces required to complete exocytosis(5-7). We find that actin and non-muscle myosin II (NMII) assemble into previously undescribed polyhedral-like lattices around the vesicle membrane. The NMII lattice comprises bipolar minifilaments(8-10 )as well as non-canonical three-legged configurations. Using photobleaching and pharmacological perturbations in vivo, we show that actomyosin contractility and actin polymerization together push on the underlying vesicle membrane to overcome the energy barrier and complete exocytosis(7). Our imaging approach thus unveils a force-generating actomyosin lattice that regulates secretion in the exocrine organs of live animals.

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.

Neutral Re(i) tetrazolato complexes exhibit labeling of lipid droplets with high specificity.

a School of Pharmacy and Medical Science, University of South Australia, Adelaide 5001 SA, Australia b Department of Chemistry, Curtin University, Bentley 6102 WA, Australia c Department of Physics and Astronomy, Macquarie University, North Ryde 2109 NSW, Australia d Department of Industrial Chemistry, University of Bologna, Bologna 40126, Italy e Centre for Microscopy, Characterisation and Analysis, University of Western Australia, Crawley 6009 WA, Australia

An effective innate immune response is critical for the protection of an organism against pathogen and environmental challenge. There is emerging evidence that an effective immune response depends heavily on the traffic and function of endosomes and lysosomes. However, there is very little understanding of the dynamics of an innate immune response, especially in vivo. Toward this aim, we have used two-photon microscopy to visualize the response to bacterial infection of the endosome-lysosome system in immune response tissues using intact Drosophila larvae. First, we set up the conditions to image intact larva in vivo and more specifically GFP-labeled endosomes-lysosomes in the fat body, and compared their distribution and size with those in tissue explanted ex vivo. Notably, we observed significant expansion of both Rab5 and Rab7 endosomal compartments upon both tissue isolation and minor aseptic wounding, indicating significant differences between live and explanted tissue. We also observed changes in endosome-lysosome vesicles within internal immune response tissues following in vivo bacterial infection by the oral route (to avoid a wounding response). We conclude that there are significant changes to the architecture of endosomes and lysosomes during an innate immune response, setting the scene for mechanistic studies to identify the signaling pathways that orchestrate this process.

Intensive cancer chemotherapy leads to significant bone loss, the underlying mechanism of which remains unclear. The objective of this study was to elucidate mechanisms for effect of the commonly used anti‐metabolite methotrexate (MTX) on osteocytes and on general bone homeostasis. The current study in juvenile rats showed that MTX chemotherapy caused a 4.3‐fold increase in the number of apoptotic osteocytes in tibial metaphysis, which was accompanied by a 1.8‐fold increase in the number of tartrate‐resistant acid phosphatase‐positive bone resorbing osteoclasts, and a 35% loss of trabecular bone. This was associated with an increase in transcription of the osteoclastogenic cytokines IL‐6 (10‐fold) and IL‐11 (2‐fold). Moreover, the metaphyseal bone of MTX‐treated animals exhibited a 37.6% increase in the total number of osteocytes, along with 4.9‐fold higher expression of the DMP‐1 transcript. In cultured osteocyte‐like MLO‐Y4 cells, MTX treatment significantly increased caspase‐3‐mediated apoptosis, which was accompanied by the formation of plasma membrane‐born apoptotic bodies and an increase in IL‐6 (24‐fold) and IL‐11 (29‐fold) mRNA expression. Conditioned media derived from MTX‐treated MLO‐Y4 cells was twice as strong as untreated media in its capacity to induce osteoclast formation in primary bone marrow osteoclast precursors. Thus, our in vivo and in vitro data suggested that MTX‐induced apoptosis of osteocytes caused higher recruitment of DMP‐1 positive osteocytes and increased osteoclast formation, which could contribute towards the loss of bone homeostasis in vivo. J. Cell. Physiol. 227: 2889–2897, 2012. © 2011 Wiley Periodicals, Inc.

The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

Osteocyte apoptosis precedes osteoclast resorption, and may act as a critical signal to trigger bone remodeling. While osteoclast precursors are known to travel via the circulation, the specific mechanisms by which they accumulate at remodeling sites are unclear. We hypothesized that osteocyte apoptosis mediates osteoclast precursor adhesion to vascular endothelium by regulating osteocytic secretion of IL-6 and soluble IL-6 receptor (sIL-6R) to promote endothelial ICAM-1 expression. We found that conditioned media from TNF-α-induced apoptotic MLO-Y4 osteocytes promoted RAW264.7 osteoclast precursor adhesion onto D4T endothelial cells (P < 0.05). Blocking osteocyte apoptosis with a pan-caspase inhibitor (ZVAD-FMK) reduced osteoclast precursor adhesion to baseline levels (P < 0.001). Endothelial cells treated with apoptotic osteocyte conditioned media had elevated surface expression of ICAM-1 (P < 0.05), and blocking ICAM-1 abolished apoptosis-induced osteoclast precursor adhesion. Apoptotic osteocyte conditioned media contained more IL-6 (P < 0.05) and sIL-6R (P < 0.05) than non-apoptotic osteocyte conditioned media. When added exogenously, both IL-6 and sIL-6R were required for endothelial activation, and blocking IL-6 reduced apoptosis-induced osteoclast precursor adhesion to baseline levels (P < 0.05). Therefore, we conclude that osteocyte apoptosis can promote osteoclast precursor adhesion to endothelial cells via ICAM-1; this is likely through increased osteocytic IL-6 and sIL-6R secretion, both of which are indispensible to endothelial activation.