SUMMARY Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.
Although infection with vaccinia virus (VV) is known to affect the cytoskeleton, it is not known how this affects the cellular architecture or whether the attenuated modified VV ankara (MVA) behaves similar to wild-type VV (wtVV). In the present study, we therefore compared effects of wtVV and MVA infection on the cellular architecture. WtVV-infection induces cell rounding early in infection, which coincides with the retraction of microtubules (MTs) and intermediate filaments from the cellular periphery, whereas mitochondria and late endosomes cluster around the nucleus. Nocodazole treatment demonstrates that cell rounding and organelle clustering require intact MTs. At the onset of virus assembly late in infection, cells reflatten, a process that coincides with the regrowth of MTs into the cellular periphery. We find that the actin network undergoes several rearrangements that occur sequentially in time and that closely follow the cell-shape changes. Unexpectedly, these actin changes are blocked or reversed upon nocodazole treatment, indicating that intact MTs are also responsible for the wtVV-induced actin rearrangements. Finally, MVA infection does not induce any of these cellular changes. Because this virus lacks a substantial number of VV genes, MVA opens up a system to search for the molecules involved in wtVV-induced cellular changes; in particular, those that may regulate actin/MT interactions.Key words: actin, cell shape, microtubules, modified vaccinia virus ankara, vaccinia virus. Vaccinia virus (VV) is the prototype of the poxviridae, a family of large DNA viruses. Its genome of about 200 kB encodes for more than 250 proteins (1) enabling VV to carry out DNA replication and transcription in the cytoplasm rather than in the cellular nucleus (1). VV is characterized by a complex cytoplasmic life cycle, directed by three classes of genes, early, intermediate and late. Following entry, the viral core is delivered into the cytoplasm, from which a defined set of early mRNAs is transcribed and extruded into the cytoplasm for translation. The early proteins are required to uncoat the viral core and to initiate cytoplasmic DNA replication. Replication sets off the transcription of intermediate and late genes, late proteins being required for virion assembly. During assembly, late in infection, two infectious forms are made; the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). A small percentage of the intracellular IMVs becomes enwrapped by a double membrane derived from the transGolgi network (TGN) or endosomes to form the intracellular enveloped virus [IEV (2,3)]. The IEV moves along microtubules (MTs) towards the plasma membrane using the plusend directed motor protein kinesin-1. Upon fusion of the outer IEV membrane with the plasma membrane, the EEV is released into the extracellular environment. The outer IEV membrane that is fused with the plasma membrane is able to polymerize actin tails, resulting in long filopodia that propel attached EEVs towards neighbouring cells, a pr...
A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFPbased fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila.Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.
SUMMARYE-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion.Recent studies identified additional E-cadherin-independent roles of E-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of E-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of E-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of E-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of E-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and E-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and E-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.
Summary Fluorescence resonance energy transfer-based executioner caspase reporters using GFP are important tools for imaging apoptosis. While these reporters are useful in imaging apoptosis in cultured cells, their in vivo application has been handicapped by poor signal to noise. Here we report design and characterization of a GFP-based fluorogenic protease reporter, dubbed “ZipGFP”. ZipGFP-based TEV protease reporter increased fluorescence 10-fold after activation by protease. A ZipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. Thus, the ZipGFP-based caspase reporter may be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond the executioner caspases.
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