ABSTRACT
Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state), that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V), previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC) resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1) protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq) is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity in photocycle kinetics among photoreceptors may be viewed as reflecting adaptive responses to specific and salient tasks required by organisms to respond to different photic environments.
Subject(s)
Adaptation, Physiological/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Light , Neurospora crassa/genetics , Photoreceptors, Microbial/genetics , Transcription Factors/metabolism , Circadian Clocks , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Genetic Loci , Genotype , Neurospora crassa/metabolism , Photoreceptors, Microbial/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/geneticsABSTRACT
Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from â¼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.
Subject(s)
Circadian Clocks , Gene Expression Regulation, Fungal , Neurospora crassa/genetics , Transcriptome/genetics , Energy Metabolism/genetics , Feedback, Physiological , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal/genetics , High-Throughput Nucleotide Sequencing , Neurospora crassa/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA , Signal Transduction/geneticsABSTRACT
Light plays an important role for most organisms on this planet, serving either as a source of energy or information for the adaptation of biological processes to specific times of day. The fungal kingdom is estimated to contain well over a million species, possibly 10-fold more, and it is estimated that a majority of the fungi respond to light, eliciting changes in several physiological characteristics including pathogenesis, development and secondary metabolism. Two model organisms for photobiological studies have taken centre-stage over the last few decades--Neurospora crassa and Aspergillus nidulans. In this review, we will first discuss our understanding of the light response in N. crassa, about which the most is known, and will then juxtapose N. crassa with A. nidulans, which, as will be described below, provides an excellent template for understanding photosensory cross-talk. Finally, we will end with a commentary on the variability of the light response among other relevant fungi, and how our molecular understanding in the aforementioned model organisms still provides a strong base for dissecting light responses in such species.
Subject(s)
Aspergillus nidulans/physiology , Gene Expression Regulation, Fungal/physiology , Neurospora crassa/physiology , Phototropism/physiology , DNA, Fungal/genetics , LightABSTRACT
The presence of disseminated tumour cells (DTCs) in bone marrow is predictive of poor metastasis-free survival of patients with breast cancer with localized disease. DTCs persist in distant tissues despite systemic administration of adjuvant chemotherapy. Many assume that this is because the majority of DTCs are quiescent. Here, we challenge this notion and provide evidence that the microenvironment of DTCs protects them from chemotherapy, independent of cell cycle status. We show that chemoresistant DTCs occupy the perivascular niche (PVN) of distant tissues, where they are protected from therapy by vascular endothelium. Inhibiting integrin-mediated interactions between DTCs and the PVN, driven partly by endothelial-derived von Willebrand factor and vascular cell adhesion molecule 1, sensitizes DTCs to chemotherapy. Importantly, chemosensitization is achieved without inducing DTC proliferation or exacerbating chemotherapy-associated toxicities, and ultimately results in prevention of bone metastasis. This suggests that prefacing adjuvant therapy with integrin inhibitors is a viable clinical strategy to eradicate DTCs and prevent metastasis.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Blood Vessels/drug effects , Mammary Neoplasms, Experimental/drug therapy , Tumor Microenvironment/drug effects , Animals , Blood Vessels/metabolism , Blood Vessels/pathology , Cell Adhesion/drug effects , Cell Line, Tumor , Cyclophosphamide/administration & dosage , Doxorubicin/administration & dosage , Female , Integrins/metabolism , Mammary Neoplasms, Experimental/blood supply , Mammary Neoplasms, Experimental/pathology , Mice, Inbred BALB C , Mice, Transgenic , Paclitaxel/administration & dosageABSTRACT
Tumor cells leave the primary tumor and enter the circulation. Once there, they are called circulating tumor cells (CTCs). A fraction of CTCs are capable of entering distant sites and persisting as disseminated tumor cells (DTCs). An even smaller fraction of DTCs are capable of progressing toward metastases. It is known that the DTC microenvironment plays an important role in sustaining their survival, regulating their growth, and conferring resistance to therapy. But we still have much to learn about the nature of these rare cell populations to predict which will progress and what exactly should cause concern for future relapse. Although recent technological advances in our ability to detect and molecularly and functionally characterize CTCs and DTCs promise to unravel this ambiguity, the timing of dissemination and the precise source of CTCs and DTCs profiled will impact the conclusions that can be made from these endeavors. In this review, we discuss the biology of CTCs and DTCs; the technologies to detect, isolate, and profile these cells; and the exceptions we must apply to our understanding of what role these cells play in the metastatic process. We conclude that a greater effort to understand the unique biology of these cells in context will positively impact our ability to use these cells to predict outcome, monitor treatment efficacy, and reveal therapeutically relevant targets to deplete these populations and ultimately prevent metastasis.
Subject(s)
Neoplasm Metastasis/pathology , Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Animals , Blood Platelets/metabolism , Blood Platelets/pathology , Humans , Neoplasm Invasiveness/pathology , Neoplasms/metabolism , Neoplastic Cells, Circulating/metabolism , Tumor MicroenvironmentABSTRACT
Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes.