Anal. Chem. 2003, 75, 2123-2130
Analysis of Individual Acidic Organelles by Capillary Electrophoresis with Laser-Induced Fluorescence Detection Facilitated by the Endocytosis of Fluorescently Labeled Microspheres Kathryn M. Fuller and Edgar A. Arriaga*
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
Submicrometer-sized fluorescent microspheres were loaded into the acidic organelles of NS-1 mouse myeloma cells via endocytosis. Confocal microscopy imaging showed that microspheres colocalized nearly perfectly with LysoTracker Red, a probe that stains acidic organelles. Unlike LysoTracker dyes that seem to leak from acidic organelles upon cell disruption, microspheres are retained within these organelles, facilitating their analysis following isolation. Using capillary electrophoresis (CE) with laserinduced fluorescence detection (LIF), the electrophoretic mobilities of acidic organelles were individually calculated and fluorescence intensities individually measured. When cells were incubated for sufficient time to allow for endocytosis (48 h) with 3.9 × 103 microspheres/cell, replicate CE-LIF analyses of the corresponding isolated fraction indicated a dramatic increase in the number of detected events (n ) 1990 ( 234) and in the overall fluorescence intensity of the individual events (0.38 ( 0.01 RFU; average ( SD; n ) 3) over the corresponding 290. When taking into account the mobilities reported here and the Booth-Overbeek theory,46 this κR value strongly suggests that the electrophoretic mobility of acidic organelles is a mainly a function of surface charge density and not size; a strong correlation between the individual migration time and fluorescence intensity measurements would be indicative of a strong influence of size on the measured electrophoretic mobility and this was not observed. Indeed, this is a simplified description that needs to be confirmed by future studies describing the effect of parameters that affect electrophoretic mobility of colloidal particles such as pH, ionic strength, and buffer composition on the organelle mobility. Furthermore, the results reported here have not taken into account any residual interactions between the capillary wall and the acidic organelle. These interactions may (46) Radko, S. P.; Stastna, M.; Chrambach, A. Anal. Chem. 2000, 72, 59555960.
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result in lower mobility values and could bias the interpretation of the data presented here. On the other hand, it is worth comparing the measured mobilities for different organelles isolated from NS-1 cell cultures: the median mobility reported here for acidic organelles and previously reported for mitochondria29 are -(1.79 ( 0.01) × 10-4 (N ) 1990 ( 234; Table 1) and -(1.90 ( 0.02) × 10-4 cm2 V-1 s-1 (N ) 43 ( 10), respectively. This comparison points to utility of electrophoretic mobility as indicator of organelle surface composition and to the possibility of using CE-LIF for the separation of acidic organelles from mitochondria. CONCLUSION The work described herein demonstrates the use of submicrometer-sized fluorescent microspheres to fluorescently label acidic organelles in NS-1 cells that are isolated and then analyzed by CE-LIF. Unlike other organelles that can be directly labeled with probes in solution, the direct treatment of cells with lysomotropic fluorescent probes in solution is not compatible with the analysis of isolated acidic organelles because of leakage from the organelles prior to CE-LIF. Since individual microspheres
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could not be detected individually, the detection of acidic organelles suggests that fusion of acidic organelles is taking place. This is the first report of an in vitro approach to analyzing individual isolated acidic organelles, and the wide range of fluorescent intensities seen substantiates the value of probing individual organelles. This technique could be used to separate multiple types of fluorescently tagged organelles or to study endocytosis in biologically relevant systems. ACKNOWLEDGMENT The authors are grateful to Dr. Richard Walsh for assistance with confocal microscopy experiments and Mr. Brent Seager for coating capillaries. K.M.F. acknowledges initial support from a National Institute of Health Chemistry/Biology Interface Training Grant (Grant GM08700). This work is supported by the National Institute of Health (Grant R01 GM61969).
Received for review December 31, 2002. Accepted February 27, 2003. AC026476D
