Digital Fluorescence Imaging of Trafficking of Endosomes Containing Low-Density Lipoprotein in Brain Astroglial Cells 1

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1 Bochemcal and Bophyscal Research Communcatons 269, (2000) do: /bbrc , avalable onlne at on Dgtal Fluorescence Imagng of Traffckng of Endosomes Contanng Low-Densty Lpoproten n Bran Astroglal Cells 1 Tomomtsu Ichkawa,* Makoto Yamada, Dasaku Homma,* Rchard J. Cherry, Ian E. G. Morrson, and Suguru Kawato*,2 *Department of Bophyscs and Lfe Scences, Graduate School of Arts and Scences, Unversty of Tokyo at Komaba 3-8-1, Meguro, Tokyo 153, Japan; Department of Physcs, School of Medcne, Kyorn Unversty, Mtaka, Tokyo 181, Japan; and Department of Bologcal Scences, Unversty of Essex, Central Campus, Colchester CO4 3SQ, Unted Kngdom Receved January 12, 2000 We have used dgtal fluorescence mcroscopy to examne transport of LDL-contanng endosomes n rat bran astroglal cells to show that ndvdual mddle endosomes undergo rapd transtons between forward/backward movements and mmoble states over short dstances. The populaton of rapdly movng endosomes (>0.04 m/sec) was 35.9%, and the remanng endosomes were slowly movng or temporarly mmoble (<0.04 m/sec). The averaged moton was, however, a very slow pernuclear moton wth a velocty of 3.25 m/h. Ths small velocty s manly due to frequent changng of drectons n movements, requrng6hfor a sgnfcant concentraton around the crcumference of the cell nucle. The applcaton of both ant-dynen antbodes and vanadate n permeablzed cells resulted n perpherally concentrated dstrbuton of endosomes, probably due to nhbton of pernuclear moton by dynen-lke motor protens. These results mply that both dynen-lke and knesn-lke protens bnd to the same endosome resultng n both pernuclear and perpherally drected movements Academc Press Astroglal cells gude neuronal development, metabolze ons and neurotransmtters, and regulate central nervous system vasculature (1). Neurosterod synthess s observed n neuroglal cells such as astrocytes and olgodendrocytes (2, 3). Neurosterods are demonstrated Abbrevatons used: cytochrome P450scc, cytochrome P450 havng cholesterol sde-chan cleavage actvty (P45011A1); DI, doctadecyl tetramethylndocarbocyamne perchlorate; LDL, low densty lpoproten; MEM, Dulbecco s modfed Eagle s medum. 1 Ths work s supported by grants from the Mnstry of Educaton, Scence and Culture n Japan and from BBSRC n the UK. 2 To whom correspondence should be addressed. Fax: E-mal: kawato@phys.c.u-tokyo.ac.jp. to acutely modulate neurotransmtter receptors and thereby to affect neuron neuron communcatons. We have recently reported the uptake of low densty lpoproten (LDL) and the exstence of neurosterodogenc protens n astrocytes (4). Neurosterod synthess may occur by hydroxylaton of sterods by cytochrome P450. Cholesterol s utlzed n mtochondra where cytochrome P450scc cleaves a sde-chan of cholesterol, resultng n pregnenolone producton. Pregnenolone would be transformed nto pregnenolone sulfate whch modulates the NMDA subtype of glutamate receptors (5). LDL s a carrer of cholesterol and cholesterol esters whch are substrates for sterodogeness. LDL s ncorporated nto endosomes va LDL receptor-medated endocytoss (6). In rat astrocytes, LDL s endocytosed, and then delvered to lysosomes where LDL molecules are dgested (7). Free cholesterol may be then transported to mtochondra. Also free cholesterol may be transferred to cholesterol ester and stored n the cytoplasm untl used. There are several reports whch suggest that LDL may be suppled from blood to astrocytes. LDL was shown to be transcytosed across the blood-bran barrer (8). The trace amount of LDL was observed n cerebrospnal flud (9). The receptor of LDL was suggested to partcpate n cholesterol uptake of glal cells (10). There has been lttle real-tme analyss of LDL-contanng endosome traffcs n ndvdual glal cells. We employed dgtal fluorescence mcroscope and sngle endosome trackng analyss to clarfy molecular mechansm of ntracellular endosome transport n the tme scale of mnutes to hours. EXPERIMENTAL PROCEDURES Chemcals. Doctadecyl tetramethylndocarbocyanne perchlorate (DI) was purchased from Molecular Probes (Eugene, OR); acrdne orange, Sodum orthovanadate, and saponn were from Wako (Tokyo, Japan). Monoclonal ant-dynen mouse IgG, clone No. 70.1, was purchased from Sgma X/00 $35.00 Copyrght 2000 by Academc Press All rghts of reproducton n any form reserved.

2 Preparaton of astroglal cell cultures. Prmary bran cell cultures were prepared from male Wstar rat embryo E20 as prevously descrbed. After a fnal centrfugaton, the cell pellet was resuspended ( cells/ml) n MEM soluton contanng 5% calf serum, 0.4% glucose, 0.3% NaHCO 3, 292 g/ml glutamne, and 100 g/ml kanamycn n a tssue culture dsh. After 2 4 weeks of culture, when confluence had been attaned, the cultured cells were trypsnzed, dspersed n MEM soluton, and replated at a densty of cells/ml. The culture medum was renewed on the thrd day of the culture. For mcroscopc observaton, cells were plated onto 35-mm-dameter glass-bottom dshes prevously coated wth poly-l-lysne. The cells were cultured for 4 5 days at a cell densty of cells/cm 2. The purty of the cultured astrocytes were determned by mmunostanng of the cells wth antbody aganst glal fbrllary acdc proten (GFAP), showng more than 95% of cells were GFAP postve. Isolaton and labelng of LDL wth DI. LDL was solated from fresh bovne blood by the modfed method of Havel et al. (11). DI was dssolved n DMSO, and 400 l of the soluton (2 mm) was ncubated wth mg/ml of LDL n a total volume of 3 ml at room temperature for 2 h. Large DI aggregates were removed by fltraton of solutons wth 0.20 m of pore sze. Unbound free DI was removed from labeled LDL by mcrocentrfugaton through a Sephadex G-25 column. Fluorescence magng and analyss. We used a vdeo-enhanced fluorescence mcroscope equpped wth a SIT camera (Hamamatsu Photoncs C-1145, Japan) and wth the temperature chamber whch mantaned the ar atmosphere at 37 C. For DI-LDL, we used nm for exctaton and fluorescence above 590 nm was selected wth flters and DM580 dchroc mrror. Ol mmerson objectve lens of 60 was used for sngle endosome trackng analyss, and dry objectve lens of 40 was used for all other experments. The vdeo output was dgtzed and the mages were stored n frame memory. Data acquston and mage analyss were performed wth ARGUS-50 system (Hamamatsu Photoncs, Japan). Sngle endosome trackng analyss. The procedures requred to track fluorescent partcles through tme-lapse mages have been gven n detal elsewhere (12). The endosomes contanng DI-LDL appeared as spots coverng a number of pxels wth dameter of m; ther approxmate postons were dentfed by a smple mage analyss algorthm, and then quantfed by least-squares fttng the pxels n ths mmedate area wth a 2-dmensonal Gaussan functon. After quantfcaton of the spots, they were lnked through the tme-lapse mages by a nearest spot wth smlar ntensty probablty method. We assgned ndvdual tracks of endosome movement to be drected motons wth retrograde motons along the mcrotubules. Ths analyss was based on the followng expermental evdences whch support the mddle endosome movements along mcrotubule networks usng dynen-lke and knesn-lke motor protens: (a) Depolymerzaton of mcrotubules by the presence of nocodazole completely nhbted the endosome movement. (b) Specfc nhbtors of dynen motors consderably changed the endosome movement. In ths case of drected moton, the dsplacement R k and the velocty vector V k of the th endosome were calculated at tme k t usng kth and (k 1)th mages, where the r k s a poston vector of the th endosome n kth mage and t s the tme nterval between mages usng 1.4 sec. R k 2 x k t t x k t 2 y k t t y k t 2 V k r k 1 r k / t The mnus end of mcrotubules anchors n the centrosome close to the nucleus and the plus end of mcrotubules s postoned around cell perpherals. Because mcrotubules have curved structure between these two ends, the drectons of mcrotubules was often not [I] [II] parallel to the straght vector from the cell perpheral to the nucleus. For quanttatve analyss of pernuclear moton of endosomes, we calculated the pernuclear velocty Vpn k and perpherally drected velocty Vph k usng the nner products: Vpn k V k, W, V k, W 0 Vph k V k, W, V k, W 0 [III] [III ] where W s a unt bass vector from the ntal poston of the -th endosome to the center of cell nucleus. RESULTS Pernuclear Movement and Concentraton of Mddle Endosomes The formaton of mddle endosomes wth mn dameter appeared about mn after the 10-mn of pulse LDL addton. At mn after the LDL addton, the mddle endosomes were dstrbuted almost unformly over the entre cytoplasmc space (Fg. 1). The dameter of mddle endosomes estmated from the varance of 2D-Gausan ftted fluorescent spots was dstrbuted as follows: between m (11.7%), between m (86.5%), and between m (1.8%). Each mddle endosome contnuously showed saltatory motons contanng forward/backward motons and mmoble states over the tme range from 1hto6hafter the LDL addton. The mddle endosomes showed very slow pernuclear movement, resultng n a concentraton of the mddle endosomes around the crcumference of nucle at 6 h after the LDL addton. To quantfy the degree of endosome concentraton around the nucleus, we evaluated the rato of fluorescent ntensty of the doughnuttype dstnct dense fluorescent regon at mmedately outsde of the nucleus, havng a band wdth equal to the radus of nucleus, to the total cytoplasmc fluorescent ntensty n ndvdual cells. The degree of endosome concentraton was found to be % at 1 h, % at 3 h and % at 6 h after the LDL addton. These concentrated endosomes were late endosomes havng acdc ph around 5, because they showed strong fluorescence wth acrdne orange stanng. Mddle endosomes, whch undergo pernuclear movement before the concentraton around nucle, were not staned wth acrdne orange. To dentfy mddle endosomes and late endosomes, cells were doubly fluorescent staned wth the DI-labeled LDL and acrdne orange. However, we could not dstngush between late endosomes and lysosomes both of whch were acdc wth ph 5 and manly localzed around the nucle. Effect of Depolymerzaton of Mcrotubules by Nocodazole To examne the possblty of endosome sldng on the mcrotubule networks, we depolymerzed mcrotubules by nocodazole. After the prencubaton of cells wth 10 26

3 cells showed no sgnfcant concentraton of endosomes around the crcumference of nucle even at 3, 6 and 12 h after the LDL addton, however, the mddle endosome formaton was observed at 1 h after the LDL addton. When nocodazole was removed by replacng the outer medum wth the Hepes medum, a sgnfcant concentraton of endosomes around the nucle was then observed at 6 h after the nocodazole depleton. These results mply that endosomes undergo movement along mcrotubule networks. We also examned the effect of actn flament dsrupton by applyng 100 nm cytocharasn D, at 15 mn before the LDL addton or at 90 mn after the LDL addton. In cytocharasn D-prencubated cells, formaton of mddle endosomes was nhbted. When cytocharasn D was appled at 90 mn after the LDL addton, we found no dfference n the mddle endosome formaton and ther concentraton around nucle n comparson wth the control cells. These results suggest that actn mcroflaments are nvolved n the formaton of mddle endosome but do not partcpate n ther movements n the cytosol. Modulaton of Dynen by Ant-Dynen Antbody and Vanadate FIG. 1. Fluorescence mages of the dstrbuton of endosomes n astrocytes. Cells were ncubated for 1 h (a), 3 h (b), and 6 h (c) at 37 C after the pulse addton of DI-LDL. The crcumferences of cells are ndcated wth whte dotted lnes. The scale s ndcated wth a horzontal black bar. M nocodazole for 3 h, DI-LDL was added and endosome dstrbuton was observed at 1, 3, 6 and 12 h later than the nocodazole treatment. Nocodazole-treated We nvestgated the possble contrbuton of dynenlke protens and knesn-lke protens whch may carry endosomes along mcrotubule networks. For nhbton of dynen-lke motor protens, cells were permeablzed wth 0.05% saponn, and ncubated for 15 mn wth ant-dynen antbody (clone 70.1) at 1:100 dluton n modfed lyss buffer contanng 1 mm ATP (13). The antbody was added at 90 mn after the pulsed LDL addton. The presence of ant-dynen antbody prevented the concentraton of endosomes around nucle, resultng n the appearance of endosomes around the cell perpheral at 4 h after LDL addton (Fg. 2). We also appled low dose of vanadate to modulate dynen-lke motor protens. Cells were ncubated wth the lyss buffer contanng 10, and 50 M freshly prepared vanadate as a substtute for dynen antbody. We observed perpheral dstrbuton of the LDL endosomes under these vanadate condtons wth no sgnfcant concentraton of endosomes around the crcumference of nucle. Ths perpherally-drected moton of endosomes was observed n less than 50 M vanadate but the formaton of mddle endosomes was never nhbted. In these condtons, we checked mcrotubule dstrbuton by Cy2 mmunostanng. Our nterpretaton of these results s that nhbton of dynenlke protens by ether antbody or vanadate prevents pernuclear moton and causes facltated antpernuclear moton nduced by knesn-lke motor protens. It should be noted that accordng to ths nterpretaton, a mddle endosome contans must contan both dynen and knesn. 27

4 Drected Moton of Indvdual Endosomes wth a Perod of Retrograde Motons along the Mcrotubule Networks We performed sngle endosome trackng analyss n order to nvestgate the detaled moton of ndvdual endosomes. Fgure 3 shows typcal trajectores of mddle endosomes n astroglal cells at 3 h after LDL addton over a tme of 84 sec. Endosomes exhbted three dfferent types of movement along the mcrotubules: forward moton, backward moton, and an mmoble state. Many of these apparently mmoble endosomes began to undergo rapd movement after a perod of several mnutes, mplyng that they were not permanently mmoble. Conversely some of rapdly movng endosomes became mmoble for a perod of tme when we traced them over several mnutes. Thus endosomes appear to swtch between moble and mmoble states. FIG. 2. Effect of antbody aganst dynen on the endosome movement. (a) Dstrbuton of endosomes n control cells at 6 h after the pulse addton of LDL. Note that cells were treated by the same procedures as (b) wth lyss buffer except ant-dynen IgG. (b) Dstrbuton of endosomes n the presence of ant-dynen antbody at 4 h after the LDL addton. Ant-dynen IgG was ncorporated nto cells at 90 mn after the LDL addton. FIG. 3. Trajectores of ndvdual endosomes n astrocytes and ther velocty dstrbuton. (A) Trajectores of mddle endosomes over 84 sec from 60 successve mages. These mages were taken at 3 h after the pulse LDL addton. The edge of the nucleus of the cell s placed n the bottom-rght corner. (B) Velocty dstrbuton of the mddle endosomes. Veloctes are averaged over 84 sec for each endosomes. The postve and negatve values correspond to the velocty of pernuclear moton, Vpn, and perpherally drected moton, Vph, respectvely. The dstrbuton was analyzed at 3 h after the LDL addton. Data were obtaned from 32 cells n several tens of ndependent experments. Error bar means SE. 28

5 A quanttatve analyss of pernuclear drected motons was performed by calculatng the pernuclear velocty, Vpn, and perpherally-drected velocty, Vph. Fgure 3 shows hstograms of the dstrbuton of Vpn (postve horzontal axs) and Vph (negatve horzontal axs) whch were averaged over 84 sec for each endosomes at 3 h after the LDL addton. The quanttatve analyss showed that the populaton of rapdly movng endosomes ( 0.04 m/sec) was 35.9% at 3 h after the LDL addton. The remanng endosomes were slowly movng or temporary mmoble wth a velocty slower than 0.04 m/sec. Most of the rapdly movng endosomes were transported bdrectonally (forward and backward) wth a velocty of m/sec (32.3%). Less than 3.6% of endosomes had a rate greater than 0.24 m/sec at 3 h after the LDL addton. The 51% of endosomes that undergo pernuclear moton slghtly exceed the endosome populaton of 49% havng perpherallydrected moton at 3 h after the LDL addton. When many tracks of endosomes were averaged, the averaged moton showed very slow pernuclear moton wth a velocty of 3.25 m/h. Untl endosomes reached the crcumference of the nucle, they move over 20 m needng almost 6htogoacross from the cell perphery, as a sum of forward, backward and mmoble movements. Once endosomes became located around the nucle, these large endosomes became permanently mmoble even n the tme scale of hours. Although the real endosome movement s 3-dmensonal, we performed the 2-dmensonal approxmaton for the analyss of endosome movement because of the followng consderatons: Because the thckness of the cytoplasmc space was smaller than 1 m determned wth confocal mcroscopy, the contrbuton of the vertcal movement could be less than 1/20 of the possble horzontal movement of 20 m (half dameter of the cell). Around the nucle havng the thckness of 2 3 m, endosomes dd not move consderably not only horzontally but also vertcally, therefor they do not sgnfcantly contrbute to the vertcal movement. DISCUSSION We focused on the analyss of the ntracellular transport of the mddle endosomes. The mddle endosomes carry a long dstant transport of cholesterol across the cytoplasmc space, whle the movements of early and late endosomes were relatvely small n dstance. Dynen antbodes caused perpherally drected moton of mddle endosomes due to nhbton of dynen-lke motors. The selectve bndng of antdynen IgG to astrocyte dynen-lke proten was demonstrated by Western mmunoblottng, showng the sngle mmunoreactve band at about 70 kda whch mght be a dynen ntermedate chan. Ant-dynen antbody (70.1) was reported to nhbt cytoplasmc dynen n cultured cod melanophores, resultng n the pgment movement toward the cell perphery (14). We also demonstrated that a low-dose of vanadate at M selectvely nhbted dynen-lke motors, nducng perpherally-drected moton of endosomes. In melanophores, 50 M of vanadate selectvely nhbted retrograde moton, nducng melanosomes dsperson caused by antretrograde-drected knesn bound to the same melanosomes (14). The requrement of hgher concentratons of vanadate (10 50 M) for dynen nhbton than those needed for nhbton of the Mg-ATPase actvty of purfed cytoplasmc dynen (around 1 M) may be due to the exstence of many ATP bndng stes n ntact cells other than dynens (15). The above results can be nterpreted as ndcatng that both dynen- and knesn-lke protens bnd to same endosomes. If only dynen motors bnd to endosomes, perpherally drected moton of endosomes cannot be nduced by the presence of dynen antbody. If only knesn motors bnd to endosomes, generally pernuclear moton could not occur. Ths vew of endosomes contanng both dynen and knesn motors s consstent wth our observaton that mddle endosomes frequently changed drecton of moton between forward and backward drectons. Many endosomes that are mmoble over a short tme range of several mnutes are probably detached from mcrotubules. Although ndvdual endosomes undergo rapd forward/backward movements and mmoble states on a short tme scale, on a long tme scale these dfferent movements of endosomes are averaged, resultng n the very slow pernuclear moton. The results lead to a novel quanttatve pcture of cholesterol transport n glal cells. REFERENCES 1. Walz, W. (1995) n Receptors of Physology n Neurogla (Ketterman, H., and Ranson, B. R., Eds.), pp Oxford Unversty Press, New York. 2. Bauleu, E. E., and Robel, P. (1990) J. Sterod Bochem. Mol. Bol. 37, Mellon, S. H., and Deschepper, C. F. (1993) Bran Res. 629, Kmoto, T., Asou, H., Ohta, Y., Muka, H., Chernogolov, A. A., and Kawato, S. (1997) J. Pharm. Bomed. Anal. 15, Wu, F. S., Gbbs, T. T., and Farb, D. H. (1991) Mol. Pharmacol. 40, Kovanen, P. T., Faust, J. R., Brown, M. S., and Goldsten, J. L. (1979) Endocrnology 104, Gullaume, D., Bertrand, P., Dea, D., Davgnon, J., and Porer, J. (1996) J. Neurochem. 66, Dehouck, B., Fenart, L., Dehouck, M. P., Perce, A., Torper, G., and Cecchell, R. (1997) J. Cell Bol. 138,

6 9. Salen, G., Bergner, V., Shore, V., Horak, I., Horak, E., Tnt, G. S., and Shefer, S. (1987) N. Engl. J. Med. 316, Jung-Testas, I., Wentraub, H., Dupus, D., Eychenne, B., Bauleu, E. E., and Robel, P. (1992) J. Sterod. Bochem. Mol. Bol. 42, Havel, R. J., Eder, H. A., and Bragdon, J. H. (1955) J. Cln. Invest. 34, Wlson, K. M., Morrson, I. E., Smth, P. R., Fernandez, N., and Cherry, R. J. (1996) J. Cell. Sc. 109, Grundstrom. N., Karlsson, J. O., and Andersson, R. G. (1985) Acta Physol. Scand. 125, Nlsson, H., and Walln, M. (1997) Cell. Motl. Cytoskeleton 38, Hsanaga, S., and Saka, H. (1983) J. Bochem. 93,