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<P>Vergne <I>et al</I>. 10.1073/pnas.0409716102.</TD>
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<H2>Supporting Information</H2>
<H4>Files in this Data Supplement:</H4>
<A HREF="#F1">Supporting Text</A><BR>
<A HREF="#F2">Supporting Movie 1</A><BR>
<A HREF="#F3">Supporting Movie 2</A><BR>
<A HREF="#F4">Supporting Movie 3</A><BR>
<A HREF="#F5">Supporting Movie 4</A><BR>
<A HREF="#F6">Supporting Movie 5</A><BR>
<A HREF="#F7">Supporting Figure 6</A><BR>
<A HREF="#F8">Supporting Figure 7</A><BR>
<A HREF="#F9">Supporting Figure 8</A><BR>
<A HREF="#F10">Supporting Figure 9</A><BR>
<A HREF="#F11">Supporting Figure 10</A><BR>
<BR><BR><BR><BR>
<A NAME="F1"></A>
<B><P>Supporting Text</P>
</B>
<B><P>Four-Dimensional (4D) Confocal Microscopy. </B>Live cell imaging was performed on a rotating disk confocal microscope (Ultra View LCI, PerkinElmer) affording low-photobleaching and low-phototoxicity long-term observations of cells. Transfected RAW 264.7 cells were synchronously infected by centrifugation of bacteria onto macrophages adherent to coverslips at 1,000 rpm for 5 min. Coverslips were mounted into a perfusion chamber (Harvard Apparatus) set at 37<FONT FACE="Symbol">°</FONT>
C. Mycobacterial entry was identified as previously published (1). Serial confocal sections (1.8 <FONT FACE="Symbol">m</FONT>
m) within a <I>z</I>-stack spanning a total thickness of 15 <FONT FACE="Symbol">m</FONT>
m were taken to ensure that all sides of the phagocytosed particles were examined for each time point regardless of object movement in or out of any given single plane of focus. For quantification, <I>z</I>-stacks were collapsed into a single <I>x</I>-<I>y</I> projection as previously described for 4D microscopy analyses (1-3). To correct for any photobleaching (minimized by the use of UltraView), cytosolic fluorescence was subtracted from that of the phagosomal membranes.</P>
<P>1. Chua, J. & Deretic, V. (2004) <I>J. Biol. Chem.</I> <B>279,</B> 36982-36992.</P>
<P>2. Gerlich, D., Beaudouin, J., Gebhard, M., Ellenberg, J. & Eils, R. (2001) <I>Nat. Cell Biol.</I> <B>3,</B> 852-855.</P>
<P>3. Gerlich, D. & Ellenberg, J. (2003) <I>Nat. Cell Biol.</I>, Suppl., S14-S19.</P>
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<A href="{__picrender__}pnas_102_11_4033__09716Movie1.mov">Supporting Movie 1</A>
<A NAME="F2"></A>
<B><P>Movie 1.</B> PI3P persists on phagosomes with dead BCG. RAW 264.7 cells were transfected with P40PX-EGFP (green) and allowed to phagocytose Texas red-labeled BCG (red). Frames were taken 93 s apart. The movie is played at 2 fps.</P>
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<A href="{__picrender__}pnas_102_11_4033__09716Movie2.mov">Supporting Movie 2</A>
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<B><P>Movie 2.</B> P40PX-EGFP is not recruited to phagosomes harboring live BCG. RAW 264.7 cells were transfected with P40PX-EGFP (green) and allowed to phagocytose Texas red-labeled live BCG (red). Frames were taken 51 s apart. The movie is played at 3 fps.</P>
<BR><BR><BR><BR>
<A href="{__picrender__}pnas_102_11_4033__09716Movie3.mov">Supporting Movie 3</A>
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<B><P>Movie 3.</B> PI3K inhibitor LY294002 washout restores PI3P on phagosomes containing dead BCG. RAW 264.7 cells were transfected with P40PX-EGFP (green) and allowed to phagocytose Texas red-labeled BCG (red). Cells were treated with a reversible <A NAME="CJSmark"></A>phosphatidylinositol 3-kinase (PI3K) inhibitor (+LY294002). Washout of the inhibitor (marked with the word washout) allows recruitment of the PI3P probe to the phagosomal membrane. Frames were taken 101 s apart. The movie is played at 2 fps.</P>
<BR><BR><BR><BR>
<A href="{__picrender__}pnas_102_11_4033__09716Movie4.mov">Supporting Movie 4</A>
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<B><P>Movie 4.</B> MTM1-EGFP is recruited to dead BCG phagosomes immediately after entry and detaches permanently within minutes. RAW 264.7 cells were transfected with MTM1-EGFP (green) and allowed to phagocytose Texas red-labeled BCG (red). Frames were taken 60 s apart. The movie is played at 2 fps.</P>
<BR><BR><BR><BR>
<A href="{__picrender__}pnas_102_11_4033__09716Movie5.mov">Supporting Movie 5</A>
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<B><P>Movie 5.</B> MTMR3-EGFP is recruited to dead BCG phagosomes immediately after entry and desorbs permanently within minutes. RAW 264.7 cells were transfected with MTMR3-EGFP (green) and allowed to phagocytose Texas red-labeled BCG (red). Frames were taken 60 s apart. The movie is played at 2 fps.</P>
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<A href="{__picrender__}pnas_102_11_4033__1.pdf">Supporting Figure 6</A>
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<B><P>Fig. 6.</B> Cytosolic Ca<SUP>2+</SUP> increase can only transiently restore phosphatidylinositol 3-phosphate (PI3P) levels on phagosomes harboring live mycobacetria. RAW 264.7 cells were transfected with P40PX-EGFP, a probe specific for PI3P (1, 2), although also reported to bind moesin in a phosphatidylinositol-dependent manner (3). The macrophages were allowed to phagocytose dead or live <I>Mycobacterium tuberculosis</I> var. <I>bovis</I> bacillus Calmette�Guérin (BCG) (labeled with Texas red). (<I>Insets</I>) Grayscale images of green (<I>Left</I>) and red (<I>Right</I>) fluorescence of areas with objects indicated by arrows. Main panels represent merged green and red fluorescence images. Treatment with Ca<SUP>2+</SUP> ionophore A23187 was carried out as described (4, 5). (<I>A</I>-<I>C</I>) Persistence of PI3P probe on dead BCG phagosomes. (<I>D</I>-<I>F</I>) Live BCG phagosomes do not recruit the PI3P probe. (<I>G</I>-<I>I</I>) Treatment with A23187 transiently restores PI3P generation on live BCG phagosomes. (<I>J</I>) Temporal analysis and quantification [relative fluorescence unit (RFU)] of GFP fluorescence on live BCG phagosomes upon A23187 treatment (filled triangles) vs. untreated cells (open triangles). (<I>K</I>) [Ca<SUP>2+</SUP>]<SUB>c</SUB> rise in RAW cells (mean of 60 cells) upon addition of 1 <FONT FACE="Symbol">m</FONT>
M A23187. (<I>L</I> and <I>M</I>) Ratio (340/380 nm) images before (<I>L</I>) or after (<I>M</I>) addition of 1 <FONT FACE="Symbol">m</FONT>
M A23187. Color-coded 340/380 nm ratio look-up table: red, highest Ca<SUP>2+</SUP>; dark blue, lowest Ca<SUP>2+</SUP>. Ca<SUP>2+</SUP> imaging and ratiometric analysis were performed using a TILL Photonics (Planegg, Germany) system. The following methods were used for ratiometric [Ca<SUP>2+</SUP>]<SUB>c</SUB> imaging. RAW 264.7 cells were loaded with 5 <FONT FACE="Symbol">m</FONT>
M fura-2-acetoxy-methyl ester in Hanks' balanced salt solution for 30 min at 37<FONT FACE="Symbol">°</FONT>
C. Ratiometric [Ca<SUP>2+</SUP>]<SUB>c</SUB> imaging was performed using an IX70 Olympus microscope equipped with a TILL Ca<SUP>2+</SUP> imaging system. Fura-2 loaded cells were illuminated at 340 nm for 150 ms and 380 nm for 50 ms in 1-s intervals using a TILL Polychrome monochromator. 340/380 fluorescence ratios (emission at 510 nm) were generated using TILL software, as described (6).</P>
<P>1. Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., Cantley, L. C. & Yaffe, M. B. (2001) <I>Nat. Cell Biol.</I> <B>3,</B> 675-678.</P>
<P>2. Lemmon, M. A. (2003) <I>Traffic</I> <B>4,</B> 201-213.</P>
<P>3. Wientjes, F. B., Reeves, E. P., Soskic, V., Furthmayr, H. & Segal, A. W. (2001) <I>Biochem. Biophys. Res. Commun.</I> <B>289,</B> 382-388.</P>
<P>4. Malik, Z. A., Denning, G. M. & Kusner, D. J. (2000) <I>J. Exp. Med.</I> <B>191,</B> 287-302.</P>
<P>5. Malik, Z. A., Thompson, C. R., Hashimi, S., Porter, B., Iyer, S. S. & Kusner, D. J. (2003) <I>J. Immunol.</I> <B>170,</B> 2811-2815.</P>
<P>6. Vergne, I., Chua, J. & Deretic, V. (2003) <I>J. Exp. Med.</I> <B>198,</B> 653-659.</P>
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<A href="{__picrender__}pnas_102_11_4033__2.pdf">Supporting Figure 7</A>
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<B><P>Fig. 7.</B> Recruitment of myotubularin (MTM) 1 and MTMR3 during phagocytosis cannot account for PI3P differences on phagosomes with dead vs. live <I>M. tuberculosis</I> var. <I>bovis</I> BCG. RAW 264.7 cells were transfected with MTM1-EGFP or MTMR3-EGFP, were allowed to phagocytose dead or live BCG (Texas red-labeled), and were examined by 4D microscopy using an UltraView system. MTM1 is recruited with a similar kinetics to live (<I>A</I>-<I>C</I>) and dead (<I>D</I>-<I>F</I>) BCG phagosomes during and immediately after phagocytosis. MTMR3 is recruited with a similar kinetics to live (<I>G</I>-<I>I</I>) and dead (<I>J</I>-<I>L</I>) BCG phagosomes during and immediately after phagocytosis. (<I>Left</I>) Red fluorescence of BCG (grayscale). (<I>Center</I>) Green fluorescence of MTM1-GFP or MTMR3-GFP (grayscale rendition). (<I>Right</I>) Merged green and red channel images. (<I>M</I> and <I>N</I>) Temporal analysis and quantification of GFP fluorescence expressed as percent-fraction of the maximum relative fluorescence intensity (RFU) of MTM1 (<I>M</I>) or MTMR3 (<I>N</I>) on dead BCG phagosomes (filled squares) or live BCG phagosomes (open triangles).</P>
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<A href="{__picrender__}pnas_102_11_4033__3.pdf">Supporting Figure 8</A>
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<B><P>Fig. 8.</B> Inhibition of protein synthesis in <I>M. tuberculosis</I> var. <I>bovis</I> BCG by the bacteriostatic antibiotic chloramphenicol causes reversible increase in PI3P positivity of phagosomes harboring live mycobacteria. RAW 264.7 cells were transfected with P40PX-EGFP and allowed to phagocytose: live, Texas red-labeled BCG (<I>A</I>), live BCG grown in the presence of chloramphenicol (+CM) (<I>B</I>) , or live BCG treated with chloramphenicol followed by drug washout (CM WO) (<I>C</I>). Images were taken and analyzed by 4D microscopy using an UltraView microscope. (<I>D</I>) Fraction (%) of PI3P-positive phagosomes. The data presented in <I>A</I>-<I>D</I> were collected at 50 min post-phagocytosis. (<I>E</I>) Optical density of BCG culture grown in the absence (filled squares) or presence (open squares) of chloramphenicol. Arrows indicate addition of chlormaphenicol (CM) and commencement of the drug washout (CM WO).</P>
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<A href="{__picrender__}pnas_102_11_4033__4.pdf">Supporting Figure 9</A>
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<B><P>Fig. 9.</B> Inhibition profile of <I>M. tuberculosis</I> SapM PI3P phosphatase activity matches that of <I>M. tuberculosis</I> culture filtrate proptein (MtCFP). Standard malachite green phosphatase quantification assay was carried out with PI3P, 50 <FONT FACE="Symbol">m</FONT>
g/ml MtCFP (<I>A</I>) or 2 <FONT FACE="Symbol">m</FONT>
g/ml of purified SapM (<I>B</I>), in the presence of indicated compounds. Note the exquisite sensitivity of the PI3P phosphatase activity to molybdate and ZnCl<SUB>2</SUB> in both <I>A</I> and <I>B</I> sets. Error bars represent the sampling error.</P>
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<A href="{__picrender__}pnas_102_11_4033__5.pdf">Supporting Figure 10</A>
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<B><P>Fig. 10.</B> Molybdate, a membrane-impermeant inhibitor of SapM, promotes maturation of phagosomes with live mycobacteria <I>in vivo</I>. RAW 264.7 cells were infected with live BCG (<I>A</I>-<I>D</I>), dead BCG (<I>E</I>-<I>H</I>), or live BCG treated with 1 mM sodium molybdate (<I>I</I>-<I>L</I>), chased for 1 h, fixed, permeabilized, and immunostained for CD63. (<I>M</I>) Fraction (%) of live BCG, live BCG treated with sodium molybdate, or dead BCG phagosomes that colocalize with the late endosomal marker, CD63. **, <I>P</I> < 0.0001 compared with live BCG phagosomes.</P>