Stimulation of the Gαq/phospholipase Cß1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cß1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCß1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCß1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCß1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCß1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

Re-track: Software to analyze the retraction and protrusion velocities of neurites, filopodia and other structures.

We have developed new software, Re-track, that will quantify the rates of retraction and protrusion of structures emanating from the central core of a cell, such as neurites or filopodia. Re-Track, uses time-lapse images of cells in TIFF format and calculates the velocity of retraction or protrusion of a selected structure. The software uses a flexible moving boundary and has the ability to correct this boundary throughout analysis. Re-Track is fast, platform independent, and user friendly, and it can be used to follow biological events such as changes in neuronal connections, tip-growing cells such as moss, adaptive migration of cells, and similar behavior in non-biological systems.

Gαq-mediated calcium dynamics and membrane tension modulate neurite plasticity.

The formation and disruption of synaptic connections during development are a fundamental step in neural circuit formation. Subneuronal structures such as neurites are known to be sensitive to the level of spontaneous neuronal activity, but the specifics of how neurotransmitter-induced calcium activity regulates neurite homeostasis are not yet fully understood. In response to stimulation by neurotransmitters such as acetylcholine, calcium responses in cells are mediated by the Gαq/phospholipase Cß (PLCß)/phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) signaling pathway. Here, we show that prolonged Gαq stimulation results in the retraction of neurites in PC12 cells and the rupture of neuronal synapses by modulating membrane tension. To understand the underlying cause, we dissected the behavior of individual components of the Gαq/PLCß/PI(4,5)P2 pathway during retraction and correlated these with the retraction of the membrane and cytoskeletal elements impacted by calcium signaling. We developed a mathematical model that combines biochemical signaling with membrane tension and cytoskeletal mechanics to show how signaling events are coupled to retraction velocity, membrane tension, and actin dynamics. The coupling between calcium and neurite retraction is shown to be operative in the Caenorhabditis elegans nervous system. This study uncovers a novel mechanochemical connection between Gαq/PLCß /PI(4,5)P2 that couples calcium responses with neural plasticity.

Regulation of bifunctional proteins in cells: Lessons from the phospholipase Cß/G protein pathway.

Some proteins can serve multiple functions depending on different cellular conditions. An example of a bifunctional protein is inositide-specific mammalian phospholipase Cß (PLCß). PLCß is activated by G proteins in response to hormones and neurotransmitters to increase intracellular calcium. Recently, alternate cellular function(s) of PLCß have become uncovered. However, the conditions that allow these different functions to be operative are unclear. Like many mammalian proteins, PLCß has a conserved catalytic core along with several regulatory domains. These domains modulate the intensity and duration of calcium signals in response to external sensory information, and allow this enzyme to inhibit protein translation in a noncatalytic manner. In this review, we first describe PLCß's cellular functions and regulation of the switching between these functions, and then discuss the thermodynamic considerations that offer insight into how cells manage multiple and competitive associations allowing them to rapidly shift between functional states.