Academic journal article Genetics

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis Elegans Motor Neurons

Academic journal article Genetics

A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis Elegans Motor Neurons

Article excerpt

BOTH neurons and neuroendocrine cells rely on the controlled release of neuropeptides via dense core vesicle (DCV) exocytosis to evoke or modulate behaviors (Scheller and Axel 1984; Kupfermann 1991; T. Liu et al. 2007; Li and Kim 2008). The DCVs in neuroendocrine and PC12 cells are much more abundant and accessible to biochemical and physiological experiments than those in neurons. For example, in chromaffin and pancreatic b-cells (both neuroendocrine cells), DCVs can number in the tens of thousands per cell and can occupy 31 and 12% of the cell volume, respectively (Dean 1973; Plattner et al. 1997). Exploiting these advantages, studies in PC12 and neuroendocrine cells have revealed that DCVs arise from a regulated secretory pathway. The pathway begins in the trans Golgi, where various sorting mechanisms cause regulated secretory proteins, such as neuropeptides and their processing enzymes, to coalesce into vesicles that bud from the trans Golgi to form immature DCVs. Additional sorting of non-DCV cargos away from DCV cargos occurs as DCVs mature through this pathway (Arvan and Castle 1998; Tooze et al. 2001; Borgonovo et al. 2006).

DCVs must selectively retain and protect several distinct cargos that have different physical states as they mature. These include the neuropeptide core, which is thought to be in an aggregated state, the neuropeptide-processing enzymes PC-2 convertase and carboxypeptidase E, possibly soluble cargos, and transmembrane cargos.

While neurons and neuroendocrine cells share this core pathway for DCV production, neurons have evolved additional membrane-trafficking requirements that may necessitate modifications to this core pathway or additional levels of regulation. For example, neurons send most of their DCVs to the axon as opposed to amassing them in the cell soma as neuroendocrine cells do. As neuronal DCVs complete their maturation in the cell soma, but before they are transported to the axon, neurons may need a mechanism to prevent the loss of those newly formed DCVs due to exocytosis from the cell soma in response to electrical depolarization or chemical signals impinging on the soma.

The model organism Caenorhabditis elegans has several strengths for investigating neuronal DCV trafficking, including the ability to track and quantitatively image DCV cargos in live animals using fluorescently tagged cargos expressed from integrated transgenes, and the ability to perform large forward genetic screens to uncover the molecular requirements for DCV trafficking. Past studies used these strengths to show that null mutations in UNC-108 (Rab2) cause altered interactions between immature DCVs and early endosomes, resulting in the loss of soluble and transmembrane cargos without affecting the aggregated neuropeptide core (Edwards et al. 2009; Sumakovic et al. 2009).

In the current study, we performed a forward genetic screen aimed at finding other mutations that alter the distribution of DCVs and DCV cargos between cell somas and axons. From the screen we recovered loss-of-function (nonsense) mutations in UNC-43 (CaM kinase II) that reduce the axonal levels of DCVs and all DCV cargos examined by °90% while cell soma/dendrite levels of all nontransmembrane cargos were reduced by °60-80%. In contrast, small synaptic vesicles were largely unaffected. Our analysis of DCV distribution, movements, and exocytosis in unc-43 mutants revealed a surprising new function for CaM kinase II in blocking the regulated exocytosis of DCVs before they are transported into axons.

Materials and Methods

Worm culture and strains

Worm culture and manipulation essentially followed previously described methods (Brenner 1974; Stiernagle 2006). Briefly, culture media was modified NGM (referred to as NGM-LOB), containing no added calcium or magnesium, and consisted of the following (per liter): 2 g NaCl, 3.1 g peptone, 3.0 g KH2PO4, 0.5 g K2HPO4, and 20 g Sigma A- 7002 agar. After autoclaving and cooling to 55°, the following was added (per liter): 1. …

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