The present experiments were designed to obtain information on the distribution and dynamics of the cellular PtdIns pools in living mammalian cells. Since there is no known protein domain that would specifically recognize PtdIns, we approached this question from two directions. First, we used a PtdIns-specific bacterial PLC to hydrolyze PtdIns and captured the reaction product, DAG with a sensitive DAG sensor. Second, we used GFP-tagged enzymes of PtdIns synthesis, PIS and the CDS1 and -2. Both of these approaches identified highly mobile small particles in the cytoplasm as the sites of PtdIns synthesis and the source of DAG when a PtdIns-specific bacterial PI-PLC is expressed in the cytosol. The PIS positive membranes originate from the ER and use an exit pathway that requires the cycling of the small GTPase Sar1. These organelles have lighter density than the bulk of ER and they contain little if any CDS1/2, the enzyme that supplies CDP-DAG for PtdIns synthesis.
The relationship between the DAG positive structures in PI-PLC expressing cells and the PIS positive organelles is not clear at present. Co-localization studies in live cells were uninformative because of the speed of movements of these structures relative to the speed of image acquisition and because fixation distorted the appearance of DAG positive particles. The DAG vesicles are more numerous and many of them are smaller than the PIS organelles and hence, likely to be at least partially distinct from the latter. However, this could simply be due to higher number of DAG sensors being associated with these objects making their detection more efficient. Nevertheless, the connection between these structures is strongly indicated by the ability of the ER- or PM-targeted PI-PLC to decrease the number of DAG particles. We also cannot rule out the possibility that the tiny DAG positive particles originate from the PIS positive membranes formed by the action of the expressed PI-PLC enzyme.
A few observations suggest that the mobile fraction of the PIS enzyme is functionally different from the one associated with the tubular ER. First, the highest specific activity of the PIS enzyme was associated with light membrane fractions separated from the bulk ER. Second, a catalytically inactive PIS enzyme did not get sorted in the mobile compartment. Third, membrane fractionation from cells expressing both PIS-HA and Sar1-H79G, which prevents the formation of the dynamic PIS pool, greatly reduced the PIS activity associated with the light membrane fractions. Notably, Imai and Gershengorn (1987)
found significantly higher Km
values for myo
-inositol and CDP-DAG for the PIS activity present in the ER membranes than in the membrane fractions that they attributed to the PM. It is not unlikely that the PIS in their “PM” fraction corresponded to the mobile compartment described in the present study. More studies are needed to understand the functional difference between the two pools of PIS enzymes.
Although the nature of these objects is still being evaluated, these findings have altered our views on PtdIns synthesis and distribution. These dynamic, mobile PtdIns distribution platforms would represent a very efficient way of making multiple contacts and deliver PtdIns to the various membranes. Dynamic contact zones between the ER and PM have been increasingly recognized as special sites for lipid transfer and metabolism (Stefan et al., 2011
). The current findings indicate that a special PtdIns rich dynamic organelle potentially multiplies the probability of making contacts and exchange lipids with a variety of membranes. A possible way of delivering lipids from the mobile platforms to the target membranes would be a simple “fusion” event but we were unable to detect fusions with the PM or other organelles, although such fusions likely occur with the ER. We also ruled out by photoactivation experiments that the PIS positive membranes would simple be ER-derived transport vesicles directed to the Golgi. While these experiments did not rule out that PtdIns is distributed by simple vesicular transport, it is equally possible that lipid transfer proteins such as the PITPs participate in lipid exchange during the brief contacts between the PIS organelle and other membranes. We have not yet been able to identify the PITP(s) that would serve in that capacity. Since CDP-DAG synthesis is mostly confined to the ER proper, the PIS positive organelles must also return to the ER to resupply the PIS enzyme with its substrate and the multiple contacts and likely fusion events observed between the tubular ER and the PIS organelle could provide the basis for such an exchange.
The PtdIns specific PI-PLC also allowed us to selectively deplete PtdIns in the PM and the ER after targeting the enzyme to these membranes. Both of these manipulations depleted the small cytoplasmic DAG positive particles and also decreased the size of myo-[3H]-labeled PtdIns4P pools and eliminated the localization of PtdIns4P and PtdIns3P recognizing protein modules. These results suggested that PtdIns equilibrates between membranes during the prolonged times (16–24 hrs) of transfection. However, there was a significant difference between the effects of ER- or PM-targeted PI-PLC on PtdIns(4,5)P2. Not surprisingly, PtdIns(4,5)P2 was largely depleted when PtdIns was consumed at the PM. However, somewhat unexpectedly, PtdIns depletion via the ER only moderately decreased PM PtdIns(4,5)P2 under unstimulated conditions. This result suggested that a significant fraction of PtdIns in the ER PtdIns pools is not directed toward the PM and that PM PtdIns were likely originated directly from the PIS organelle. In this context it is important to note that during PIS knockdown there was a parallel decrease in PtdIns, PtdIns4P and PtdIns(4,5)P2 indicating that once PtdIns synthesis is intercepted at the source, it will equally affect all phosphoinositide pools.
There are several open questions that remain to be answered in future studies. How is the generation of the PIS organelle regulated and how does the cells sense the PtdIns status of its membranes? Our preliminary studies showed no obvious change in PIS distribution after agonist stimulation when PtdIns resynthesis is increased, or after loading cells with DAG analogues. It is most likely that we need a thorough quantitative analysis of the statistical behavior of these organelles. The frequency and resident times of contacts probably hold the key to answer these questions, but clearly more studies are needed to fully explore every dimension of these processes.
In summary, the present studies introduced several approaches to determine the localization of PtdIns in living cells and to manipulate PtdIns levels in distinct cellular compartments. Our results revealed that a significant part of PtdIns synthesis is associated with a dynamic ER-derived structure that is capable of making multiple contacts with a variety of organelles and is highly suitable to supply PtdIns lipids to the different membranes. We propose a separate name of PIPEROsome (PI Producing ER-derived Organelle) to designate this structure if it stands the scrutiny of further studies. These findings open new research directions to understand how cells generate and maintain their membrane lipid compositions and to explore the exact molecular events that take place during the contacts between these organelles and their target membranes.