ABSTRACT
Bacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model - referred to as the pore forming model - is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+-dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca-resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical 'loss of toxin binding'. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover.
ABSTRACT
Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.
