We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization dependent fluorescent proteins (ddFPs). This strategy has been used to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca2+ concentration dynamics, and other second messenger signaling activities.

Dimerization-dependent fluorescent proteins (ddFP) are a recently introduced class of genetically encoded reporters that can be used for the detection of protein interactions in live cells. The progenitor of this class of tools was a red fluorescent ddFP (ddRFP) derived from a homodimeric variant of Discosoma red fluorescent protein. Here, we describe the engineering and application of an expanded palette of ddFPs, which includes green (ddGFP) and yellow (ddYFP) variants. These optimized variants offer several advantages relative to ddRFP including increased in vitro contrast and brightness for ddGFP and increased brightness and a lowered pK a for ddYFP. We demonstrate that both variants are useful as biosensors for protease activity in live cells. Using the ddGFP tool, we generated a highly effective indicator of endomembrane proximity that can be used to image the mitochondria-associated membrane (MAM) interface of endoplasmic reticulum (ER) and mitochondria.

The expanding repertoire of genetically encoded biosensors constructed from variants of Aequorea victoria green fluorescent protein (GFP) enable the imaging of a variety of intracellular biochemical processes. To facilitate the imaging of multiple biosensors in a single cell, we undertook the development of a dimerization-dependent red fluorescent protein (ddRFP) that provides an alternative strategy for biosensor construction. An extensive process of rational engineering and directed protein evolution led to the discovery of a ddRFP with a K(d) of 33 μM and a 10-fold increase in fluorescence upon heterodimer formation. We demonstrate that the dimerization-dependent fluorescence of ddRFP can be used for detection of a protein-protein interaction in vitro, imaging of the reversible Ca²⁺-dependent association of calmodulin and M13 in live cells, and imaging of caspase-3 activity during apoptosis.

ARS2 is a regulator of RNA polymerase II transcript processing through its role in the maturation of distinct nuclear cap-binding complex (CBC)-controlled RNA families. In this study, we examined ARS2 domain function in transcript processing. Structural modeling based on the plant ARS2 orthologue, SERRATE, revealed 2 previously uncharacterized domains in mammalian ARS2: an N-terminal domain of unknown function (DUF3546), which is also present in SERRATE, and an RNA recognition motif (RRM) that is present in metazoan ARS2 but not in plants. Both the DUF3546 and zinc finger domain (ZnF) were required for association with microRNA and replication-dependent histone mRNA. Mutations in the ZnF disrupted interaction with FLASH, a key component in histone pre-mRNA processing. Mutations targeting the Mid domain implicated it in DROSHA interaction and microRNA biogenesis. The unstructured C terminus was required for interaction with the CBC protein CBP20, while the RRM was required for cell cycle progression and for binding to FLASH. Together, our results support a bridging model in which ARS2 plays a central role in RNA recognition and processing through multiple protein and RNA interactions. The generation of mature RNA in the nucleus is a highly coordinated process that requires distinct complexes for the biogenesis of different RNA families. For RNA polymerase II (RNAP II) transcripts, a critical step is the addition of a 7-methylguanosine (m7G) cap to the nascent transcript (1), which is subsequently bound by CBP20 and CBP80 of the nuclear cap-binding complex (CBC) (2). CBC-controlled transcripts include mRNA, microRNA (miRNA), replication-dependent histone (RDH) mRNA, small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA), each with its own unique processing requirements. Binding of the CBC to the m7G cap of these RNAs occurs cotranscriptionally, protects the transcripts from degradation, and plays a central role in recruiting the appropriate machinery for processing different RNA families (3-8). However, exactly how distinct RNA families are differentially recognized to allow for correct processing complex formation is not fully understood. Recently, a protein called ARS2 (or SRRT in humans) has been shown to be part of the CBC, and it plays an important role in the maturation of several distinct RNA families (8, 9).Clues to how ARS2 participates in recruiting different RNA processing machineries are beginning to emerge from biochemical and structural studies. ARS2 interacts directly with the assembled CBP20/80 cap complex to form a tertiary complex termed CBCA (9). One hypothesis is that ARS2 bridges the CBCA to the appropriate processing machinery by interacting with both protein and RNA elements. This hypothesis is based on studies of the ARS2 plant orthologue SERRATE in miRNA biogenesis. Both the Arg/Pro-rich N terminus and zinc finger (ZnF) domain of SERRATE are required for primary miRNA (primiRNA) binding (10, 11). Additionally, SERRATE, through its N terminus and ZnF, has been shown to bind directly to...

We describe a multi-purpose technology platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth, and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved an orange fluorescent protein biosensor, and engineered an enzyme with reduced sensitivity to its inhibitor. These three distinct protein analysis and engineering applications, each with unique assay requirements and different host organisms, highlight the flexibility and technical capabilities of our platform.

The discovery of naturally evolved fluorescent proteins and their subsequent tuning by protein engineering provided the basis for a large family of genetically encoded biosensors that report a variety of physicochemical processes occurring in living tissue. These optogenetic reporters are powerful tools for live-cell microscopy and quantitative analysis at the subcellular level. In this review, we present an overview of the transduction mechanisms that have been exploited for engineering these genetically encoded reporters. Finally, we discuss current and future efforts towards the combined use of various optogenetic actuators and reporters for simultaneously controlling and imaging the physiology of cells and tissues.

BackgroundEphrin A1 (EFNA1) is a member of the A-type ephrin family of cell surface proteins that function as ligands for the A-type Eph receptor tyrosine kinase family. In malignancy, the precise role of EFNA1 and its preferred receptor, EPHA2, is controversial. Several studies have found that EFNA1 may suppress EPHA2-mediated oncogenesis, or enhance it, depending on cell type and context. However, little is known about the conditions that influence whether EFNA1 promotes or suppresses tumorigenicity. EFNA1 exists in a soluble form as well as a glycophosphatidylinositol (GPI) membrane attached form. We investigated whether the contradictory roles of EFNA1 in malignancy might in part be related to the existence of both soluble and membrane attached forms of EFNA1 and potential differences in the manner in which they interact with EPHA2.ResultsUsing a RNAi strategy to reduce the expression of endogenous EFNA1 and EPHA2, we found that both EFNA1 and EPHA2 are required for growth of HeLa and SK-BR3 cells. The growth defects could be rescued by conditioned media from cells overexpressing soluble EFNA1. Interestingly, we found that overexpression of the membrane attached form of EFNA1 suppresses growth of HeLa cells in 3D but not 2D. Knockdown of endogenous EFNA1, or overexpression of full-length EFNA1, resulted in relocalization of EPHA2 from the cell surface to sites of cell-cell contact. Overexpression of soluble EFNA1 however resulted in more EPHA2 distributed on the cell surface, away from cell-cell contacts, and promoted the growth of HeLa cells.ConclusionsWe conclude that soluble EFNA1 is necessary for the transformation of HeLa and SK-BR3 cells and participates in the relocalization of EPHA2 away from sites of cell-cell contact during transformation.

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