confocal-microscopy-reveals-cell-surface-receptor-aggregation-through

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of ...

La Trobe University

During germination of barley grains, the cell walls of the starchy endosperm are degraded by (1-->3,1-->4)-beta-glucanases (EC 3.2.1.73) secreted from the aleurone and scutellar tissues. The complete sequence of the aleurone (1-->3,1-->4)-beta-glucanase isoenzyme II comprises 306 amino acids and was determined by sequencing nine tryptic peptides (110 residues) and aligning them with the amino acid sequence deduced from a cDNA clone encoding the 291 NH(2)-terminal residues. Although no amino acid sequence homology with a bacterial (1-->3)(1-->4)-beta-glucanase is apparent, close to 50% homology is found with two large regions of a (1-->3)-beta-glucanase from tobacco pith tissue. The gene for barley (1-->3,1-->4)-beta-glucanase isoenzyme II shares with that for the alpha-amylase isoenzyme 1 a strongly preferred use of codons with G and C in the wobble position (94% and 90%, respectively). Both enzymes are secreted from the aleurone cells during germination. Such one-sided codon usage is not characteristic for the gene encoding the (1-->3)-beta-glucanase of tobacco pith tissue or the hor2-4 gene encoding the B(1) hordein storage protein in the endosperm.

Primary structure of the (1-->3,1-->4)-beta-D-glucan 4-glucohydrolase from barley aleurone.

Tks5 (tyrosine kinase substrate with 5 SH3 domains) is an adaptor protein which cooperates with Src tyrosine kinase to promote the formation of protease-enriched, actin-based projections known as invadopodia, which are utilized by invasive cancer cells to degrade the extracellular matrix (ECM). We previously identified a Src-Tks5-Nck pathway which promotes invadopodium formation and ECM proteolysis in melanoma and breast cancer cells. We therefore sought to investigate the significance of Tks5 expression in human cancers. This was undertaken retrospectively through an immunohistochemical evaluation in tissue microarray cores and through data mining of the public database, Oncomine. Here we showed that Tks5 was expressed at higher levels in the microarray cores of breast, colon, lung and prostate cancer tissues compared to the levels in normal tissues. Importantly, mining of Oncomine datasets revealed a strong correlation between Tks5 mRNA overexpression in a number of cancers with increased metastatic events and a poorer prognosis. Collectively, these findings suggest a clinical association of Tks5 expression in human cancers. It identifies the importance for further investigations in examining the full potential of Tks5 as a relevant prognostic marker in a select number of cancers which may have implications for future targeted therapies. 

Expression of the adaptor protein Tks5 in human cancer: prognostic potential.

Tks5 is a scaffold protein and Src substrate involved in cell migration and matrix degradation through its essential role in invadosome formation and function. We have previously described that Tks5 is fundamental for zebrafish neural crest cell migration in vivo. In the present study, we sought to investigate the function of Tks5 in mammalian development by analyzing mice mutant for sh3pxd2a, the gene encoding Tks5. Homozygous disruption of the sh3pxd2a gene by gene-trapping in mouse resulted in neonatal death and the presence of a complete cleft of the secondary palate. Interestingly, embryonic fibroblasts from homozygous gene-trap sh3pxd2a mice lacked only the highest molecular weight band of the characteristic Tks5 triplet observed in protein extracts, leaving the lower molecular weight bands unaffected. This finding, together with the existence of two human Expressed Sequence Tags lacking the first 5 exons of SH3PXD2A, made us hypothesize about the presence of a second alternative transcription start site located in intron V. We performed 5'RACE on mouse fibroblasts and isolated a new transcript of the sh3pxd2a gene encoding a novel Tks5 isoform, that we named Tks5β. This novel isoform diverges from the long form of Tks5 in that it lacks the PX-domain, which confers affinity for phosphatidylinositol-3,4-bisphosphate. Instead, Tks5β has a short unique amino terminal sequence encoded by the newly discovered exon 6β; this exon includes a start codon located 29 bp from the 5'-end of exon 6. Tks5β mRNA is expressed in MEFs and all mouse adult tissues analyzed. Tks5β is a substrate for the Src tyrosine kinase and its expression is regulated through the proteasome degradation pathway. Together, these findings indicate the essentiality of the larger Tks5 isoform for correct mammalian development and the transcriptional complexity of the sh3pxd2a gene. 

Genetic disruption of the sh3pxd2a gene reveals an essential role in mouse development and the existence of a novel isoform of tks5.

Metastatic progression is the major cause of breast cancer-related mortality. By examining multiple syngeneic preclinical breast cancer models in mice lacking a functional type-I interferon receptor (Ifnar1(-/-) mice), we show that host-derived type-I interferon (IFN) signaling is a critical determinant of metastatic spread that is independent of primary tumor growth. In particular, we show that bone metastasis can be accelerated in Balb/c Ifnar1(-/-) mice bearing either 4T1 or 66cl4 orthotopic tumors and, for the first time, present data showing the development of bone metastasis in the C57Bl/6 spontaneous MMTV-PyMT-driven model of tumorigenesis. Further exploration of these results revealed that endogenous type-I IFN signaling to the host hematopoietic system is a key determinant of metastasis-free survival and critical to the responsiveness of the circulating natural killer (NK)-cell population. We find that in vivo-stimulated NK cells derived from wild-type, but not Ifnar1(-/-), mice can eliminate the 4T1 and 66cl4 breast tumor lines with varying kinetics in vitro. Together, this study indicates that the dysregulated immunity resulting from a loss of host type-I IFN signaling is sufficient to drive metastasis, and provides a rationale for targeting the endogenous type-I IFN pathway as an antimetastatic strategy.

Loss of Host Type-I IFN Signaling Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple Models of Breast Cancer.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS. 

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Progress in next-generation sequencing technologies has facilitated investigations into microbial dynamics. An important bacterium in the dairy industry is Propionibacterium freudenreichii, which is exploited to manufacture Swiss cheeses. A healthy culture of these bacteria ensures a consistent cheese with formed 'eyes' and pleasant flavour profile, and the investigation of prophages and their interactions with these bacteria could assist in the maintenance of the standard of this food product. Two bacteriophages, termed PFR1 and PFR2, were chemically induced using mitomycin C from two different dairy strains of P. freudenreichii. Both phages have identical genomes; however, PFR2 was found to contain an insertion sequence, IS204. Host range characterisation showed that PFR1 was able to form plaques on a wild type Propionibacterium acnes strain, whereas PFR2 could not. The lytic plaques observed on P. acnes were a result of PFR1 inducing the lytic cycle of a pseudolysogenic phage in P. acnes. Further investigation revealed that both PFR1 and PFR2 could infect P. acnes but not replicate. This study demonstrates the dynamic interactions between phages, which may alter their lytic capacity under certain conditions. To our knowledge, this is the first report of two phages interacting to kill their host.

Dynamic interactions between prophages induce lysis in Propionibacterium acnes.

Mitoxantrone was efficiently encapsulated within cucurbit[8]uril in a 2:1 complex where the two mitoxantrone molecules were symmetrically located through both portals of a cucurbit[8]uril cage. The novel complex facilitates increased mitoxantrone uptake in mouse breast cancer cells and decreases the toxicity of the drug in healthy mice. In an orthotopic mouse model of metastatic breast cancer the complex still maintains  anticancer activity compared to the free drug, yet provides a statistically significant increase in the survival of these mice compared to conventional mitoxantrone treatment. This new low toxicity formulation offers the possibility to increase mitoxantrone dose and thus maximize efficacy while managing the dose limiting side effects.

Encapsulation of Mitoxantrone within Cucurbit[8]uril Decreases Toxicity and Enhances Survival in a Mouse Model of Cancer.

To isolate and characterize bacteriophage lytic for the opportunistic pathogen Klebsiella oxytoca and their formulation into a range of solid dosage forms for in-vitro testing.

Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca.

Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.

Molecular characterization of the first saltwater crocodilepox virus genome sequences from the world's largest living member of the Crocodylia.

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous  and  characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g. , , ), metastatic virulence (e.g. , ), metastatic suppression (e.g. , , , , , ) or metastatic avirulence (e.g. ). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC.This article has an associated First Person interview with the first author of the paper.

Article	Year
Primary structure of the (1-->3,1-->4)-beta-D-glucan 4-glucohydrolase from barley aleurone. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 16593676	1986
Expression of the adaptor protein Tks5 in human cancer: prognostic potential. Oncology reports\| PubMed ID: 24993883	2014
Genetic disruption of the sh3pxd2a gene reveals an essential role in mouse development and the existence of a novel isoform of tks5. PloS one\| PubMed ID: 25259869	2014
Loss of Host Type-I IFN Signaling Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple Models of Breast Cancer. Cancer immunology research\| PubMed ID: 26198985	2015
Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS. Acta neuropathologica\| PubMed ID: 26298469	2015
Dynamic interactions between prophages induce lysis in Propionibacterium acnes. Research in microbiology\| PubMed ID: 27639668	2017
Encapsulation of Mitoxantrone within Cucurbit[8]uril Decreases Toxicity and Enhances Survival in a Mouse Model of Cancer. ACS medicinal chemistry letters\| PubMed ID: 28523107	2017
Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca. PloS one\| PubMed ID: 28817689	2017
Molecular characterization of the first saltwater crocodilepox virus genome sequences from the world's largest living member of the Crocodylia. Scientific reports\| PubMed ID: 29618766	2018
Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer. Disease models & mechanisms\| PubMed ID: 29720474	2018

Peter Lock

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