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Lipids are distributed in unique compositions within
the different membranes of a eukaryotic cell. Their unequal distribution
plays an essential role in a number of cell functions. Thus a major
question in cell biology is how various lipids are taken up into
and sorted within a cell to form these unique combinations. Studies
in which cells of the yeast Saccharomyces cerevisiae are grown in
the presence of phospholipids with a shortened acyl chain attached
to the 7-nitrobenz-2-oxa-1 3-diazol-4-yl (NBD) fluorophore have
shown that lipids may be internalized and sorted based on head group;
however effects of differences in the structures of the hydrophobic
side chains had not been examined. For our project we studied the
internalization of four phosphatidylcholine (PC) analogs (same head
group) with different fluorophore side chains using fluorescence
microscopy and flow cytometry to measure location and extent of
uptake. Our results suggest that hydrophobic side chains in addition
to hydrophilic head groups also play an important role in determining
the mechanism by which a lipid is taken up into a cell.
The various membranes of a eukaryotic cell contain
uniquely distributed combinations of lipids. For example an asymmetric
distribution of phospholipids exists on the inner and outer leaflets
of the plasma membrane of red blood cells the disruption of which
sets up the clotting cascade (reviewed by Hanson and Nichols 2001).
While the consequences of the unique membrane lipid compositions
of a cell are far-reaching an equally important question is how
the different lipids are internalized and sorted into their specific
combinations. Phospholipid internalization has been studied using
lipids containing a shortened hydrocarbon chain attached to a fluorescent
molecule or fluorophore. Internalization of 7-nitrobenz-2-oxa-1
3-diazol-4-yl (NBD)-tagged phospholipids in the yeast Saccharomyces
cerevisiae reveals sorting based on the structure of the hydrophilic
head group in which the addition of a single methyl group to NBD-labeled
phosphatidylethanolamine (NBD-PE) is sufficient to cause it to be
trafficked to the vacuole similarly to NBD-phosphatidylcholine (NBD-PC)
(Hanson et al. 2002). NBD-PE and NBD-PC have been hypothesized to
be taken up by protein-mediated translocation (flip) across the
plasma membrane (reviewed by Hanson and Nichols 2001). More recently
specific genes have been implicated in the internalization of NBD-tagged
phospholipids. Among these are DNF1 and DNF2 coding for ATPase proteins
found predominantly in the plasma membrane which seem to play a
role in energy-dependent phospholipids flip (Pomorski et al. 2003).
An unrelated gene LEM3 seems to control specifically the uptake
of NBD-PC as well as alkylphosphocholine drugs (Hanson et al. paper
in submission). However the phospholipids studied were labeled with
NBD and possible effects of differences in hydrophobic side chain
structure were not closely examined. We studied the possible effects
of side chain differences by tracking the internalization and localization
of phosphatidylcholine (PC) tagged with one of four different fluorophores
NBD and three somewhat more hydrophobic fluorophores containing
the Bodipy group (see figure below). From our results it would appear
that the structure of the hydrophobic side chain is also significant
in determining the mechanism by which a phospholipid is trafficked
in a cell.
We tested the internalization of the PC analogs with
several variables:
- Temperature: In all experiments cells were incubated with the
lipids both at 30 degrees C the optimal temperature and at 2 degrees
C (on ice). Low temperature blocks vesicular transport including
endocytosis a possibly significant mode of internalization.
- ATP depletion: To deplete ATP we re-suspended cells in synthetic
media containing azide with or without 10mM fluoride and incubated
them briefly at 30 degrees C before adding lipid.
- Membrane potential: Incubation with CCCP a protonophore collapses
the proton electrochemical gradient across the plasma membrane.
Cells were incubated with 10mM CCCP for 10 minutes at 30 degrees
C before labeling with lipids.
- Deletions of NBD-PC transporter genes: The strains used in
this experiment were LMY94 (wild-type LEM3) LMY102 (lem3D) LMY161
(wild-type DNF1DNF2) LMY165 (dnf1D) LMY166 (dnf2D) and LMY167
(dnf1Ddnf2D). The deletion strains have been shown to inhibit
NBD-PC uptake.
All experiments were done on overnight cultures in
complete media (YPD) grown to log phase in synthetic media (SDC).
Cells were labeled with DMSO-solubilized lipids added to a final
concentration of 5mM for either 30 minutes at 30 degrees C or 1
hour on ice. Localization of fluorescence was viewed by fluorescence
micrsocopy. Uptake was measured in terms of mean intensity of fluorescence
using flow cytometry and pixel measurements on fluorescence microscope
images.
- Fluorophore structure affects sorting of PC analogs within a
cell (Figure 1). Bodipy PC's are not trafficked to the vacuole!
- Trafficking of Bodipy 581 FL and 530 to the mitochondria and
intracellular membranes does not involve vesicular transport i.e.
is not affected by low temperature (Figure 2).
- Bodipy 581 and Bodipy 530 are not internalized by endocytosis.
Their mechanism is presumably protein-mediated flip (Figure 2).
- Endocytosis is a major internalization mechanism of Bodipy
FL uptake (Figure 2). Membrane haloes in the dnf1ƒdnf2ƒ
strain at 2°--blockage of other lipids shows up simply as
lowered fluorescence (Figure 6b). A small amount of internalization
does occur at 2°C suggesting a role for flip as well.
- Fluorophore structure affects dependence on ATP (Figure 3a).
NBD and Bodipy FL are internalized by energy-dependent mechanisms
(Figure 3b). Bodipy 530 does not use an energy-dependent mechanism
of influx; however its efflux may require ATP (Figure 3b).
- Uptake of Bodipy PC's uses proton membrane force (PMF). Fluorescence
is reduced in CCCP-treated cells at both temperatures for all
4 PC's (Figure 4b).
- NBD transport to the vacuole is dependent on both ATP and PMF
(Figure 3a 4a).
- The LEM3 pathway is not the predominant uptake mechanism for
all PC analogs (Figure 5). Deletion blocks NBD and only partly
blocks Bodipy FL. 9. DNF1 and DNF2 form a major pathway for Bodipy
FL but not for Bodipy 530 and 581 (Figure 6)
Fluorophore structure affects 1)localization of lipid and 2) mechanism
of uptake.
Future Directions
- Internalization of Bodipy lipids and PDR1 PDR3 gain of function
mutants have been shown to experience down-regulated flip and
up-regulated flop of NBD-phospholipids (Hanson and Nichols 2001).
- Bodipy FL uptake in end4ƒ mutantsóto test dependence
on endocytosis.
Lab Principal Investigator: Dr. J. Wylie Nichols
Lab Technician: Lynn Malone
Graduate Student: Haley Curtis
Post-doc: Shelley Elvington
This material is based upon work supported by the Howard Hughes
Medical Institute under Grant No. 52003727 and by the National Institutes
of Health under Grant No. R01GM64770.
Lipids are a major building block of cells and exist in different
combinations in the various membranes that make up a eukaryotic
cell. The mechanism by which they are taken up into the cell and
sorted into their target locations is a major question in cell biology.
Tracking the internalization can be done by growing cells in the
presence of lipids made fluorescent by molecules attached to their
side chains. Phospholipids are made up of a polar head group and
two nonpolar side chains. Earlier studies had suggested that the
head group in a phospholipid could be a way for cells to distinguish
them and take them up and sort them specifically. Our project was
to grow yeast cells with lipids with different fluorescent molecules
attached to their side chains to see if side chain structure could
also be a means of sorting. We looked at the localization of the
fluorescence under a microscope and measured amount of uptake by
looking at average intensity of the fluorescence using a machine
called a flow cytometer. Our results show that side chain does make
a difference in whether and how a lipid is taken up and sorted to
its eventual locations in a cell.
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