A fluorescence-based sensor for calibrated measurement of protein kinase stability in live cells.

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

A Fluorescence-Based Sensor for Calibrated Measurement of Protein Kinase Stability in Live Cells.

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We monitor the fluorescence of kinases fused to a fluorescent protein relative to that of a co-expressed reference fluorescent protein. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

New insights into Raf regulation from structural analyses.

BRAF is a highly regulated protein kinase that controls cell fate in animal cells. Recent structural analyses have revealed how active and inactive forms of BRAF bind to dimers of the scaffold protein 14-3-3. Inactive BRAF binds to 14-3-3 as a monomer and is held in an inactive conformation by interactions with ATP and the substrate kinase MEK, a striking example of enzyme inhibition by substrate binding. A change in the phosphorylation state of BRAF shifts the stoichiometry of the BRAF:14-3-3 complex from 1:2 to 2:2, resulting in stabilization of the active dimeric form of the kinase. These new findings uncover unexpected features of the regulatory mechanisms underlying Raf biology and help explain the paradoxical activation of Raf by small-molecule inhibitors.

Spatial regulation of the actin cytoskeleton by HSF-1 during aging.

There are many studies suggesting an age-associated decline in the actin cytoskeleton, and this has been adopted as common knowledge in the field of aging biology. However, a direct identification of this phenomenon in aging multicellular organisms has not been performed. Here, we express LifeAct::mRuby in a tissue-specific manner to interrogate cytoskeletal organization as a function of age. We show for the first time in Caenorhabditis elegans that the organization and morphology of the actin cytoskeleton deteriorate at advanced age in the muscles, intestine, and hypodermis. Moreover, hsf-1 is essential for regulating cytoskeletal integrity during aging, so that knockdown of hsf-1 results in premature aging of actin and its overexpression protects actin cytoskeletal integrity in the muscles, the intestine, and the hypodermis. Finally, hsf-1 overexpression in neurons alone is sufficient to protect cytoskeletal integrity in nonneuronal cells.

Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems.

Novel tools and methods for regulating in vivo plant gene expression are quickly gaining popularity and utility due to recent advances in CRISPR-dCas9 chimeric effector regulators, otherwise known as CRISPR artificial transcription factors (CRISPR-ATFs). These tools are especially useful for studying gene function and interaction within various regulatory networks. First generation CRISPR-ATFs are nuclease-deactivated (dCas9) CRISPR systems where dCas9 proteins are fused to known transcriptional activator domains (VP64) or repressor domains (SRDX). When multiple chimeric dCas9-effector fusions are guided to gene regulatory regions via CRISPR gRNAs, they can modulate expression of transcript levels in planta. The protocol presented here provides a detailed procedure for activating AtPAP1 and repressing AtCSTF64 in Arabidopsis thaliana. This protocol makes use of our plant CRISPR toolbox to streamline the assembly and cloning of multiplex CRISPR-Cas9 transcriptional regulatory constructs.

Nuclear transport dysfunction: a common theme in amyotrophic lateral sclerosis and frontotemporal dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases with overlapping genetic factors and pathology. On the cellular level, a majority of ALS and FTD cases are characterized by nuclear clearance and cytoplasmic aggregation of otherwise nuclear proteins, TAR DNA-binding protein 43 (TDP-43), or fused in sarcoma. Recent studies investigating cellular pathways perturbed by genetic risk factors for ALS/FTD converge on nucleocytoplasmic transport dysfunction as a mechanism leading to disease pathophysiology. We propose that mutations in FUS and hexanucleotide expansions in C9orf72 and aging all converge on the impairment of nucleocytoplasmic transport, which results in the hallmark pathological feature of ALS/FTD - cytoplasmic aggregation of TDP-43 or FUS.

CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects.

The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.

Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.

C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.

Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative disorders with clinical, genetic, and neuropathological overlap. Hexanucleotide (GGGGCC) repeat expansions in a noncoding region of C9ORF72 are the major genetic cause of FTD and ALS (c9FTD/ALS). The RNA structure of GGGGCC repeats renders these transcripts susceptible to an unconventional mechanism of translation-repeat-associated non-ATG (RAN) translation. Antibodies generated against putative GGGGCC repeat RAN-translated peptides (anti-C9RANT) detected high molecular weight, insoluble material in brain homogenates, and neuronal inclusions throughout the CNS of c9FTD/ALS cases. C9RANT immunoreactivity was not found in other neurodegenerative diseases, including CAG repeat disorders, or in peripheral tissues of c9FTD/ALS. The specificity of C9RANT for c9FTD/ALS is a potential biomarker for this most common cause of FTD and ALS. These findings have significant implications for treatment strategies directed at RAN-translated peptides and their aggregation and the RNA structures necessary for their production.