in vivo analysis of mt-based vesicle transport by confocal reflection microscopy

Technique Article

In Vivo Analysis of MT-Based VesicleTransport by Confocal Reflection Microscopy

Imre Gaspar and Janos Szabad*

Department of Biology, Faculty of Medicine, University of Szeged,Szeged, Hungary

The use of confocal reflection microscopy (CRM) for the in vivo analysis ofmicrotubule (MT) mediated transport of lipid droplets in the developing Drosoph-ila egg primordia is described here. The developing Drosophila oocytes are idealobjects to study MT-mediated transport in vivo: transport of e.g. the lipid dropletscan be conveniently, selectively and sensitively monitored through CRM and theegg primordia are readily available for physical, chemical and/or genetic manipu-lations. CRM is a non-destructive way to follow vesicle movement and allowshigh frame rate image recording. When combined with fluorescence imaging,CRM offers simultaneous visualization of the cargo and the protein(s) of interest,i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery.Cell Motil. Cytoskeleton 66: 68–79, 2009. ' 2009 Wiley-Liss, Inc.

Key words: confocal reflection microscopy; MT-mediated transport; vesicle transport; lipid droplet;

Drosophila oocyte

INTRODUCTION

The MTs and the associated motor molecules areimmensely involved in maintaining well coordinatedspatiotemporal distribution of the macromolecules andcell organelles in the cytoplasm [Vale, 2003]. Althoughthe elements of the transport machinery—the MTs andthe associated kinesin or dynein motors—are well char-acterized in vitro [Wang et al., 1995; Howard, 1996],their in vivo features, most importantly the coordinationof motor activity, is largely unknown. To understand theprecise control mechanism of the MT-based transport (i)one needs to follow the motion of motor-moved visiblecomponents, (ii) the process has to be amenable to mani-pulations (i.e. to small molecular inhibitors or geneticalterations) and (iii) conditions of the analysis should beas close to in vivo as possible. The developing early Dro-sophila embryo has been proposed to be a good model tostudy motor-mediated active transport as the initial stepsof embryogenesis take place in the egg cytoplasm filledwith nutritive components, including a huge amount of

lipid droplets and the system is amenable to genetic mani-pulations [Foe, 1993; Cermelli et al., 2006]. The lipiddroplets are transported along the MTs by dynein andkinesin motors and their distribution is strictly regulated

*Correspondence to: Janos Szabad; Department of Biology, Faculty

of Medicine, University of Szeged, Somogyi B. str. 4. H-6720 Szeged,

Hungary. E-mail: [email protected]

Additional Supporting Information may be found in the online version

of this article.

Contract grant sponsor: Hungarian Scientific Research Fund; Contract

grant number: OTKA NI69180; Contract grant sponsor: Graduate Stu-

dent Program of the University of Szeged.

Received 11 September 2008; Accepted 2 December 2008

Published online 6 January 2009 in Wiley InterScience (www.

interscience.wiley.com).

DOI: 10.1002/cm.20334

' 2009 Wiley-Liss, Inc.

Cell Motility and the Cytoskeleton 66: 68–79 (2009)

in a spatiotemporal fashion [Welte et al., 1998; Grosset al., 2000]. However, there are several drawbacks usingthe embryos: (i) since the studied lipid droplet transportis apico-basally directed, the embryos have to be flattenedto follow vesicle translocation. (ii) The conventionalcontrast enhancing light microscopic techniques used tovisualize lipid droplets, such as VE/DIC microscopy, aredifficult or impossible to combine with simultaneousfluorescence imaging. In the present paper, we describethe use of confocal reflection microscopy [Vesely andBoyde, 2001; Paddock, 2002] to monitor lipid dropletmotion in vivo in the developing Drosophila oocyte tocharacterize MT-based active transport. The techniqueoffers high versatility, non-destructive, high frame rateimaging of reflective objects and can be easily and freelycombined with conventional fluorescence recordings.Making use of CRM, we analyzed lipid droplet motionin the cytoplasm of the developing Drosophila oocyte andreport that while most of the particles flow along withthe kinesin heavy chain (KHC) propelled fast ooplasmicstreaming [Palacios and St Johnston, 2002; Serbus et al.,2005] there is a deviant subpopulation of the lipid drop-lets that are transported by the conventional MT-motortransport machinery. A few hundred nm long runs, fre-quent pauses and track changes characterize this type oftransport. It is also reported here that the egg primordiaare ideal objects to studyMT-based transport: their develop-ment is a well characterized process [King, 1970; Spra-dling, 1993], they are readily amenable tor physical andchemical manipulations and also to genetic alterations.

MATERIALS AND METHODS

Drosophila Stocks

To establish the CRM technique in the imaging oflipid droplet motion in the developing egg primordia, weused w1118 homozygous females as a control. (For an ex-planation of the genetic symbols see the FlyBase athttp://flybase.bio.indiana.edu.) To label the MTs, a Jupi-ter-GFP expressing protein trap line was used, a kindgift from Alain Debec. [Jupiter-GFP, - Jup-GFP forshort - is a MT associated protein with thus far unknownfunction; Karpova et al., 2006]. The Jup-GFP providesbetter contrast MT images than any of the conventionallyused Tau-GFP [Murray et al., 1998] or the Tubulin-GFPlines [Grieder et al., 2000] and does not effect viabilityand fertility perceptibly (data not shown). We recombinedJup-GFP and kavarnull onto the same chromosome. [Thekavarnull symbol stands for an X-ray induced null alleleof the aTub67C gene; Venkei and Szabad, 2005]. TheJup-GFP kavarnull/Kavar18c females descended from across between Jup-GFP kavarnull/TM3, Sb Ser femalesand Kavar18c/TM3, Sb Ser males. Kavar18c is a dominant

negative female-sterile mutation of the aTub67C gene[Erdelyi and Szabad, 1989]. It encodes the formation ofthe highly toxic E82K-a4-tubulin [Venkei and Szabad,2005; Venkei et al., 2006]. The Drosophila cultures wereraised on standard food and kept at 258C.

Preparation of the Egg Primordia

Newly hatched females were kept virgin and fedwith fresh yeast for two days when their ovaries weredissected in BRB80 buffer (80 mM PIPES, 1 mMEGTA, 2 mM MgCl2, pH 5 6.9) and transferred onto acoverslip. The egg primordia were isolated with finetungsten needles. Remnants of the buffer was carefullyremoved and replaced by 10S Voltalef halocarbon oil.The preparations were transferred for imaging onto thestage of an Olympus FV1000 microscope. To counter-stain the lipid droplets, a 12.5 lg/ml Nile Red (19123,Sigma-Aldrich) in BRB80 solution was layered onto thedissected egg primordia for 25 minutes prior to coveringthem with oil. For drug treatment, 20 ll of BRB80 con-taining 10 lg/ml colchicine (C9754, Sigma-Aldrich) waslayered on the egg primordia, which were then incubatedat room temperature for 30 minutes in a wet chamber.The excess buffer was removed following the incuba-tion, and the egg primordia were covered with 10SVoltalef oil. For cold treatment, the egg primordia werecovered with 48C halocarbon oil and transferred onto icefor 30 minutes. The samples were then transferred to amicroscope stage in a 258C air-conditioned room. Therewas no sign of damage or decay in any of the prepara-tions during the recording time of maximum 30 minutes.

Confocal Reflection Microscopy

Microscopic recordings were carried out by usingan Olympus FluoView 1000 confocal microscope. ForCRM analysis, without simultaneous detection of fluo-rescence, a BS20/80 beam splitter in the scanner unit andthe first-in-order photomultiplier (PMT) were used tominimize the loss of the reflected photons. The emissionfilter with adjustable spectrum was adjusted to a 610 nmbandwidth around the wavelength of the laser line (forexample, the emission filter was set to 623–643nm forthe 633 nm He/Ne laser line). To perform CRM analysisin combination with fluorescence, we used the 633 nmHe/Ne laser beam (with 0.6–1 mW excitation power) togenerate the reflection signal. In this setup, the BS20/80beam splitter was replaced by dichroic mirrors to providesufficiently intensive light to excite the fluorochromes.Simultaneous fluorescence imaging and detection ofreflection was achieved by using the first two PMTs forfluorescence imaging. The third PMT was combinedwith an LP560 optical filter to allow the 633 nm reflectedlight to pass to the detector. Yolk auto-fluorescence was

CRM and MT-Based Transport 69

excited by a 405 nm CO2 laser beam, and detected in the425–475 nm interval.

Image Analysis and Measurement

All measurements and analyses were carried outusing the ImageJ software (http://rsb.info.nih.gov/ij/)with the appropriate plugins. The diameter of the par-ticles was determined by measuring the area of the high-reflection signals. To estimate the number of particlesover a filed of view and measure the reflection yield ofthe different laser beams, Nile red fluorescence andreflection images were masked by the ‘‘GranulometricFilter’’ plugin [Prodanov et al., 2006] with a 0.245 lmradius, the radius of the droplets. For number estimationthe filtered particles were counted by the ‘Analyseparticles’ command on the first frame and the newlyappearing particles (e.g. by the streams) were added tothe initial value. During reflection yield measurementsthe non-droplet areas were discarded using the filteredand binarized image as an ‘AND’ mask. Then the ‘‘3DObject Counter’’ plugin provided the average intensityvalues. To determine the background reflection inten-sities, we randomly selected circular areas (with a0.245 lm radius) from the regions without droplets.

Co-localization of Nile Red fluorescence and thereflection signal was detected with the ‘‘ColocalizationThreshold’’ plugin. The colocalizing pixels and thebinarized yolk auto-fluorescence image were subtractedfrom both the Nile Red fluorescence and the 633 nmreflection images. Thus, only the non-colocalizing, non-yolk particles were left unchanged by the subtraction.These particles were counted. Number of all the particlesover a field of view was determined as described aboveon both Nile Red fluorescence and 633 nm reflectionchannels after the binarized yolk-autofluorescence wassubtracted.

Time-Lapse Imaging and Motion Analysis

High frame rate (15 fps) recording of the movingparticles was carried out using a 603, 1.35 NA UPlan-SApo oil-immersion objective with 103 zoom usingbidirectional scanning (the field of view was 20.91 3

20.91 lm2). For each egg primordium, 19.5-s longrecordings were collected over two randomly selectedareas that were a few lm away from the cortex along thez-axis. We used of the SpotTracker plugin [Sage et al.,2005] of ImageJ to follow the movement of individuallipid droplets. The follow-ups from early stage 12oocytes was clustered into four arrays depending on thetype and direction of the motion, representing: (i) ooplas-mic streaming, (ii) particles moving toward minus ends(against the stream), (iii) particles moving toward plusends faster than the stream, and (iv) a small fraction ofthe lipid droplets moving perpendicularly to the stream.

Lipid droplets that were moving (a) in the same direc-tion, (b) with seemingly the same speed as the yolk par-ticles and (c) could have been followed over long periodswere considered to move with the streams. Droplets thatmoved in the same direction but burst (could be followedfor short period and the speed exceeded the averagestream velocity 1 13SD, 458 1 156 nm/s) were clus-tered to the plus end tracking category. To test that lipiddroplets participate in all the above mentioned motionsconsidering the excellent but not 100% selectivity ofCRM for lipid droplets, we visualized them by usingHistone2Av-RFP [Schuh et al., 2007] that accumulatestransiently in the lipid droplets [Cermelli et al., 2006].The particles that were visualized by RFP fluorescenceare involved in all of the motion categories (Supp. Movie10). All the lipid droplets that belong to the non-stream-ing (ii–iv) categories were tracked. To characterizeooplasmic streaming, ten-ten streaming droplets weretracked randomly for over 150 frames each. Therecorded tracks were analyzed using Microsoft Excelwith custom made macros (see Supp. Info.). First, thelongest linear tracks were located along the path usingthe least-square fitting method with a threshold of r2 �0.95. The utilized algorithm recognizes a few short lineartracks even during the diffusion of the lipid droplets (inthe colchicine treated or E82K-a4-tubulin containingegg primordia). To avoid mixing the MT dependent dis-placement with accidental track recognition, 90th percen-tiles of the run time (in three successive frame intervals,195 ms) and the run length (178 nm) of the linear runsmeasured during diffusive movement were set as exclu-sion parameters. Analysis of the trajectiles that did notexceed both of the exclusion parameters was cancelled.The collected tracks were separated into runs by lookingfor pauses that met both of the following criteria: (i) tem-poral velocity drops below 100 nm/s and (ii) the droplast for at least two successive frame intervals (130 ms).Runs that would extend the spatial and temporal dimen-sions of the recordings were discarded. Motion of all thedroplets that exhibited movements other than streamingwas quantified.

The systematic error of the CRM technique and theapplied analytic methods was determined using earlystage 12 egg primordia fixed in 4% paraformaldehyde inPBS for 20 minutes. Then 6.5 s long (100 frames)images were collected from both the steady state (theegg primordia stood still) and from the ‘‘moving state’’specimens in which the microscope stage was moved ata constant speed along a linear path over 10 lm distancewithin 10 (high speed) and 30 s (low speed). The system-atic error of center detection was 67, 68.5, and 69 nm(average of standard deviations of the XY coordinates) at405, 515, and 633 nm wavelengths respectively, follow-ing the analysis of 20 lipid droplets over 100 frames at

70 Gaspar and Szabad

each wavelength. The systematic error of the velocitywas determined at two levels. During high speed cali-bration (the stage moved at a speed of 980 nm/s), thespeed of the fixed droplets (n 5 20) was 981 6 8 nm/s(average 6 SD) and thus the noise level was 0.82% (SD/average). At 360 nm/s stage speed, the correspondingmeasured value was 362 6 5 nm/s (n 5 20) and thus thenoise level was 1.4% for the 633 nm He/Ne laser line.

The average speed of the streaming yolk granuleswas determined by measuring the linear, end-to-enddisplacement of 40 yolk granules in four early stage 12oocytes (10 granules in each) moving along linear pathsover long intervals (>100 frames). This method gives agood, though underestimated approximation of the stateof the streaming ooplasm. The path of the yolk granulesslightly deviated from linear during the recording but themeasurement does not consider pauses (if any) duringthe movement of yolk. Thus, the real movement velocitymight be higher, similarly to the case of the lipid droplets.

Statistical analysis was carried out by one-wayANOVA test in SPSS 15. All statistical values are in pre-sented in average 6 s.e.m. form, unless otherwise noted.

RESULTS

CRM, a Novel Way to VisualizeLipid Droplet Motion

Exciting photons are reflected from the interfacebetween media with different optical densities, includingthe cellular components with large differences in refrac-tion indexes. In fluorescent microscopy, the reflectedphotons are removed by the emission filter. However,

the use of a ‘‘wrong’’ emission filter—with a transmit-tance spectrum that includes the wavelength of the excit-ing light—allows the detection of the reflected photons.While in wide-field fluorescent microscopy such setupneeds to be avoided, in confocal microscopy the pinholeaperture is an efficient tool to eliminate the overwhelm-ing background signal originating from the out-of-focusplanes (mostly form the distant surface of the coverslip)[Paddock, 2002]. When the egg primordia are analyzedwith the ‘‘reflection’’ setup in combination with a smallpinhole aperture, important details of the specimen areeasy to distinguish, such as the cell-cell boundaries, thechorion and the vitelline membranes and several types ofthe intracellular particles (Fig. 1). While the larger par-ticles are of several, the small, dot like ones are only0.49 6 0.05 lm in size (n 5 204). Size and the highnumber of the small particles suggest that they are lipidstorage vesicles, called lipid droplets. To test whetherthe small particles are indeed lipid droplets, we stainedovaries with Nile Red, a lipid droplet specific dye[Greenspan et al., 1985], and tested the co-localizationof the fluorescent and the reflection signals in the eggprimordia. The two types of signals highly - though notcompletely - overlap (Rcoloc 5 0.853 6 0.010; n 5 9)implying that the vast majority of the small particles areindeed lipid droplets (Fig. 2). Most of the non-overlap-ping signals originate from the yolk granules, which aresmall protein storage vesicles of endocytic origin[DiMario and Mahowald, 1987] and possess auto-fluo-rescence. The rest non-colocalizing, non-yolk particleswere counted: 0.7 6 0.1 % of the particles on the NileRed fluorescence channel and 2.1 6 0.3% of the

Fig. 1. Reflection imaging. Drosophila egg primordia as they appear in bright field microscope (A) and

in the 633 nm reflected light (B). Reflection imaging reveals several features of the specimen including

the detailed structure of chorion (arrow) and the vitelline membrane (note the peculiar pattern shown by

the arrowhead). Cell-cell boundaries between the nurse cells, follicle cells and the oocyte as well as the

nurse cell nuclei can be distinguished due to the numerous small, dot-like light reflecting particles that fill

the cytoplasm of the above listed cells. The objective was 203, 13 zoom. Scale bar 5 100 lm.


particles on the 633 nm reflection channel showed nooverlap, indicating that the CRM recognizes lipid drop-lets specifically (>99%) and selectively (>97.5%).

Remarkably, when the focal plane of imagingintersects the yolk granules close to the geometrical cen-ter a dot-like reflection surrounded by a dark halo is gen-

erated. This allows the yolk granules to be distinguishedfrom the lipid droplets during reflection imaging. Imag-ing the yolk granules in other focal planes reveals abroadening ring-like pattern as the function of the dis-tance from the geometrical center (Fig. 2). It appears thatthe spherical yolk granules—as well as the spherical

Fig. 2. Fluorescence and reflection imaging of particles in the oocyte

cytoplasm. The mages show yolk autofluorescence (A), Nile Red fluo-

rescence (B), and 633 nm reflection (C) of the same field of view.

Panel D shows co-localizing pixels of the Nile Red fluorescence and

the 633 nm reflection. This image was used as a mask to find non-co-

localizing signals (some non-overlapping reflection particles are indi-

cated by arrows, while non-overlapping Nile Red positive droplets by

arrowhead in panel E). In panel E, the yolk autofluorescence appears

in cyan, Nile Red fluorescence in yellow and the reflection signal in

magenta. In the reflection image (C), certain reflection properties of

the yolk granules can be recognized: (i) a dot like reflection sur-

rounded by a dark halo (dashed thin circle); (ii) a ring-like reflection

(dashed box) and (iii) inhomogenous surface reflection (solid thick

circle). The objective was 603, 53 zoom. Scale bar5 5 lm.

Fig. 3. Reflection and fluorescent imaging of a yolk granule and a

lipid droplet. The lipid droplet images appear in the inlets. The panels

show reflection (A and C) and fluorescence images of a yolk granule

and a lipid droplet (B and D). Yolk is visualized by auto-fluorescence,

the lipid droplet by Nile Red fluorescence. The A and the B panels

show Z-projected optical sections, C and D show the Z-stack rotated

around the X-axis by 908. The reflection images are based on the scat-

tered 633 nm light. While fluorescence depicts the real spherical shape

of the objects, the reflection signal appears as an hour-glass with the

thinnest and brightest part close the geometrical center of the yolk par-

ticle and the lipid droplet (C, arrows). A very likely explanation for

the observed ‘focusing’ feature of a sphere is that the light hitting the

object at a certain incident angle (in the range of a0-the critical inci-

dent angle of reflection; and a1-the maximal incident angle, where the

objective can collect the reflected light) is back-scattered (reflected to-

ward the objective). The reflected light virtually originates from a sin-

gle, ‘focus’ point of the object (E, gray area). The objective was 603,

203 zoom. Scale bars 5 1 lm in both directions.


lipid droplets - ‘focus’ the reflected light (Fig. 3).Another interesting property of many of the yolk par-ticles is that the reflection pattern is inhomogeneousclose to their surface while the emitted auto-fluorescenceappears homogeneous (see Figs. 2 and 5E).

Versatility is an attractive feature of the‘‘reflection’’ setup. The different laser lines used forreflection imaging of the live, stage 12 Drosophila eggprimordia resulted in very similar spatial information ofthe object (Fig. 4). However, mayor differences appearin the relative reflection yield and in the contrast of theimages. At identical laser power (1 mW), the 405, the543, and the 633 nm laser beams result in well saturatedimages, while the 458, the 488, and the 515 nm lines ofan Ar/Kr laser yield the lowest excitation/signal ratio. Inparallel, the signal/background ratio (the contrast)increases with longer wavelengths (Fig. 4), although

some individual reflection particles exhibit greatly dif-ferent from average reflective properties (Supp. Fig. 1).

The Nipkow disc and the laser scanning confocalmicroscope with bidirectional scanning mode allowhigh-frequency imaging, i.e. 15 frames per second (fps)in the present study. However, such scanning requires astrong excitation light to get sufficiently strong signalsthat leads to a rather fast loss of the fluorophores. In con-trast to fluorescence, a great advantage of the CRM tech-nique is that the reflection features of the particles arenot affected by photodegradation and remain constantthroughout imaging (see the Supp. Movies).

Lipid Droplets in the Developing Egg Primordia

In the developing Drosophila egg primordia, theooplasm displays a MT-mediated KHC- driven fastooplasmic streaming during stages 10B-12 [Gutzeit,

Fig. 4. Reflection efficiency of the different laser lines. The same

near-cortex area of a living early stage 12 oocyte was analyzed using

the laser lines listed on the images (in nm). Due to the very motile na-

ture of the lipid droplets, the particles can not be matched on the sub-

sequent images. Thus, a formaldehyde-fixed sample was analyzed

also (see Supp. Fig. 1.). The objective was 603, 103 zoom. Scale

bar 5 2 lm. On the graph the solid line in the right panel represents

the average signal intensities reflected from the lipid droplets. (The

number of the analyzed particles were 193, 147, 157, 178, 176, and

186 for the 405, 458, 488, 515, 543, and 633 nm laser lines, respec-

tively, in three early stage 12 oocytes.) The dashed line shows the

average background intensities (30 particle-sized background areas

were measured for each laser lines). Error bars represent standard

error of the mean. The laser power was 1 mW for each line.


1982; Serbus et al., 2005]. The MTs form loose, longparallel bundles perpendicular to the anterior-posterioraxis of the oocyte, close to the egg cortex [Januschkeet al., 2006]. Most of the cytoplasm components travelalong the bundles with a seemingly smooth movementwith no mayor changes in direction and velocity. All re-flective particles observed by CRM were motile duringthe entire recording of early stage 12 oocytes. Most ofthe lipid droplets (89.6%) travel with the stream with anaverage speed of 322 6 8 nm/s, similar to yolk granulesthat move with about 374610 nm/s (P 5 0.106; Fig. 5).

However, detailed CRM analyses of single streaminglipid droplets revealed that their actual speed is as highas 458 6 6 nm/s over about 1 sec, and is significantlyhigher than the average speed of the streaming yolkgranules (P < 0.001). Besides moving with high speedover a short time period, the streaming lipid droplets canbe monitored for relatively long periods (usually overthe entire recording). They keep short pauses or changetracks very frequently resulting in a saltatory motion(Table I). It is difficult to decide whether the particlesare moved actively by MT-mediated transport, are

Fig. 5. Motion of the lipid droplets is MT-dependent (A–F). Time

projection images (D–F)weremade by projecting 100 successive reflec-

tion images at 633 nm representing 6.5 s. The paths start in red (first

five frames) continue in yellow and finish up in green (last five frames).

Color coding was done by projecting the entire path to the blue, the

first five frames additionally to the green (cyan) and the last five frames

additionally to the red channel (purple) and subsequently inverting the

color. Standstill particles that change position neither via MTs nor by

diffusion or other means of transport appear in black. The MT images

(A–C) were prepared by taking the average of the middle ten frames

of the GFP channel of the corresponding time lapse recording. When

theMTs are present (A and D) the lipid droplets move either via stream-

ing (arrows) or by conventional MT-based transport (dashed box in D).

When the MTs were disrupted either by colchicine (B and E) or by of

E82K-a4-tubulin (C and F), the linear displacement of the lipid drop-

lets was abolished as indicated by the absence of red-yellow-green

streaks and the elevated number of standstill particles in the time pro-

jections (E and F). Time projection of the particles in the cytoplasm of

wild type early stage 12, late stage 12 and stage 13 oocytes (G–I). On

image G, the arrow indicates the direction of ooplasmic streaming. The

1, the 2, and the \-labelled lipid droplets move towar d the plus or

the minus ends of the MTs or move perpendicularly to the streaming,

respectively. Although there is no streaming in the late stage 12 oocyte,

some of the lipid droplets keep moving along linear paths (H, arrows).

There is no indication of linear particle motion in the stage 13 oocyte

(I). The objective was 603, 103 zoom. Scale bar5 2 lm.


dragged by the flowing cytoplasm or both active andpassive mechanisms contribute their motion. However,there is a small subpopulation of the lipid droplets(10.4%), which move in a different way than theooplasmic streaming (the non-streaming population).(i) Some travel in the same direction as the stream butfaster and for relatively short periods (�1 s). (ii) Othersmove perpendicularly to the stream and (iii) yet othersmove against the stream, usually with high speed.

It has been described previously that the lipiddroplets are transported along the MTs by plus endtracking kinesins and dynein motor molecules in theDrosophila embryos [Welte et al., 1998; Gross et al.,2000; 2002]. To prove the involvement of the MTs inmotion of the lipid droplets in the egg primordia, weused colchicine and E82K-a4-tubulin - encoded by theKavar18c mutant allele - to disrupt the MT tracks[Venkei et al., 2006]. Removal of the MTs broughtabout stoppage of the lipid droplet streaming. In addi-tion, there was also no sign of the fast, linear motion ofthe non-streaming lipid droplet subpopulation either inthe colchicine treated (n5 11) or in the E82K-a4-tubu-lin containing (dissected from the Kavar18c/kavarnull

females; n 5 9) early stage 12 egg primordia (Fig. 5).The present finding clearly shows the involvement ofthe MTs in the fast, non-streaming transport of the lipiddroplets.

Since the streaming is maintained by KHC, a plusend-directed motor [Palacios and St Johnston, 2002;Serbus et al., 2005], it is not too speculative to statethat in the parallel cortical MT bundles most of the MTplus ends point toward the stream, and therefore theminus ends to the opposite direction. Based on this sup-position, we grouped the oppositely moving dropletsinto plus- (the ones moving with the streaming, butfaster) and minus-end tracking groups (the ones movingagainst the streams) that are dominantly moved by plusend-directed (most likely by Kinesin I) and minus end-directed (most likely cytoplasmic dynein) motors[Hirokawa et al., 1998]. Analysis of the individual tra-jectories revealed that most of the non-streaming lipiddroplets take relatively long runs with high velocityand the runs are ended by either short pauses, trackchanges or most frequently by MT-particle dissocia-tions (Table I). Interestingly, although reversals happenthey are very infrequent (data not shown).

Toward the end of stage 12, the ooplasmicstreaming ceases and although the parallel organizationof the MTs disappear, the MTs remain intact and stillsupport fast transport of the lipid droplets but notooplasmic streaming (Fig. 5). The motion of the lipiddroplets keeps the characteristic features of the motormediated transport, yet several differences appear ascompared to the early stage 12 oocytes. (i) WithoutT

ABLEI.

ParametersofLipid

DropletTransport

inStage12DrosophilaEggPrimordia

Experim

ental

condition

Stageofegg

primordia

andtype

ofmovment

Number

ofegg

primordia

Number

ofparticles

Number

ofruns

Run

length

b

(nm)

Run

timeb

(ms)

Speedb

(nm/s)

Pause

c

Pause

durationb,c

(ms)

Lipid

droplets

affected

by

pausesd

(%)

Observed

aAnalyzed

(allin

bracket)a

Fraction

(%)

Room

temperature

Early

stage12

10

149.26

10.0

15.5

61.2

(309)

10.4

405

10346

41

11786

48

9086

15

99

1896

10

22.3

Plusende

10

149.26

10.0

6.76

0.7

(135)

4.5

177

11486

61

14446

85

8486

17

41

1886

14

22.5

Minusende

10

149.26

10.0

7.56

1.2

(150)

5.0

195

9856

58

9926

52

9756

25

47

2006

16

19.1

Streaming

5149.26

10.0

106

0(100)

89.6f

713

6446

30

13916

59

4586

6618

3326

23

100.0

Latestage12

10

144.36

13.8

7.36

1.9

(145)

5.0

202

7166

44

10266

67

7266

23

66

2136

21

30.1

Recoveryfrom

cold

treatm

entg

10min

before

574.8

65.5

12.1

62.6

(121)

16.2

171

7916

44

10766

60

7616

22

52

1836

20

26.4

5min

before

563.0

68.9

13.9

61.9

(139)

22.0

171

10346

52

12586

65

8666

25

37

3006

61

20.1

aPer

recording(20.91lm

320.91lm

319.5

s).

bAverageandstandarderrorofthemean.

cTheshortstopsandthetrackchanges

arepooled.

dPercentageofparticlefollow-upswhereatleastonepause

disruptedtherun.

eTheparam

etersarebased

onlipid

dropletsthatmoved

towardstheplusortheminusMTendsin

theearlystage12oocytecytoplasm

.f Intheearlystage12oocytesalllipid

dropletsweremovingduringtherecording.Thustheones

notbelongingto

thenon-streaminggroupareconsidered

tostream

.gThecompleterecoverytakes

15minutes,as

indicated

bytheresumed

ooplasm

icstream

ing.


ooplasmic streaming, a grouping of the motion by direc-tion was no longer possible. (ii) Although the number ofthe lipid droplets observed within the recorded area dur-ing the entire recording is not different (149.2 6 10.0 forearly stage 12 and 144.3 6 13.8 for late stage 12; P 5

0.766), the proportion of the transported lipid dropletsdecreases significantly (from 15.5 6 1.2 to 7.3 6 1.9particles/recording; P < 0.001). (iii) The speed of trans-port also decreases significantly (from 908 6 15 nm/s to726 6 23 nm/s; P < 0.001), (iv) alongside with the aver-age time spent in motion. (v) Consequently, the averagerun lengths also became shorter (Table I). The differen-ces may stem from changes in motor activation/proces-sivity or from stoppage of the ooplasmic streaming. Theflowing cytoplasm may exert viscous drag on the movingparticles, decreasing or increasing the load on themechanoenzymes. Altered load brings changes in themotor duty-cycle effecting many parameters of themotion, such as the speed of the motion, as it has beendescribed for kinesin I in in vitro studies [Coppin et al.,1997].

To elucidate the possible effects of ooplasmicstreaming on motor-mediated lipid droplet transport, weused cold treatment to temporarily stop the processes.The MTs become depolymerized during incubation onice and form again once the egg primordia are trans-ferred onto to 258C [Januschke et al., 2006]. Imaging ofone early stage 12 oocyte per preparation was done inevery minute, as described above. The recovery of theMT bundles and the restart of ooplasmic streaming inthe early stage 12 oocytes took about 15 minutes, thatwas set as the reference time (considered as complete re-covery). Ten minutes before complete recovery, the lipiddroplets started a linear, non-streaming type of motion ata significantly lower speed than without cold treatment(761 6 22 nm/s versus 908 6 15 nm/s; P < 0.001). Theaverage run time and the run length were also shorterthan in the untreated control (Table I). The motion pa-rameters were similar to those of late stage 12 untreatedegg primordia, except the number of the motile particles(12.1 6 2.6 versus 7.3 6 1.9 particles/recording for coldtreated and the late stage 12 oocytes, respectively; P 5

0.008). There was still no sign of ooplasmic streamingfive minutes before complete recovery, however featuresof lipid droplet motion were as usual: the speed was866 6 25 versus 908 6 15 nm/s (P 5 0.14), the numberof the moving particles was 13.9 6 1.9 versus 15.5 6

1.2 particles/recording (P 5 0.164) and all the othermotion parameters, such as the average run length andthe run time, became also restored (Table I). Interest-ingly, the number of all observable particles dropped toapproximately to half of the expected values (74.8 6 .5and 63.0 6 8.9 for ten and five minutes before recovery,respectively). These changes probably stem from the re-

traction of lipid droplets from the cortical areas due tocold treatment. Upon complete recovery, the number ofobservable particles started to increase (98.8 1 13.7particles/recording). The above results indicate that:(i) while the surrounding cytoplasm moves with aremarkable velocity, the ooplasmic streaming does notaffect the fast, non-streaming transport of lipid droplets.(ii) The decreasing motor activity can account for all thechanges detected in the late stage 12 oocyte, except forthe reduced number of the motile droplets.

DISCUSSION

Confocal Reflection Microscopy

There are a number of components in the livingmaterial that differ in refractive index from their sur-roundings and thus reflect light. The reflecting particlesand vesicles can be studied in light microscopes by mak-ing use of the contrast enhancing techniques such as dif-ferential interference contrast microscopy, dark fieldmicroscopy and also CRM [Vesely and Boyde, 2001;Paddock, 2002]. The advantages of all the previouslydeveloped methods are as follows: (i) they require theleast external modification in the living material andtherefore provide the most reliable information. (ii) Thesignal to be detected is not subjected to photodegradationand makes thus long-term high temporal resolutionimaging possible. (iii) The CRM technique, in addition,can be easily combined with simultaneous fluorescentimaging without any change in the instrumentation. Dueto geometrical reasons, the spherical reflective objectsseemingly focus the reflected light (Fig. 3E). Thereflected signal converges toward the center of particlesallowing thus good position determination and reliableseparation of the grouping particles during image analy-sis. In addition, many yolk granules—that are sphericaland appear homogenous by auto-fluorescence—possessinhomogeneous reflection pattern when imaged close tothe surface. Some inhomogeneity may come from rem-nants of the Golgi apparatus on the surface of the gran-ules [Giorgi and Jacob, 1977] and allows, in addition topositioning, determination of the spin of the movingyolk granules. Spin of the moving particle may provideadditional information about the nature of the motormediated transport processes, e.g. may help distinguish-ing actively carried granules (no spin) from passivelystreaming ones (the rotating motion around the threeaxes is not restricted).

The CRM technique does not require the modifi-cation of the hardware in the confocal microscope[Paddock, 2002], thus it can be established in everymicroscopic facility without any additional costs. Thesame optical elements offer full compatibility with


simultaneous fluorescence monitoring; especiallybecause any laser line (any channel) can be used forreflection detection, although with different relative sig-nal yield and contrast. However, there are certain par-ticles that have different reflective properties than themajority. These particles are probably not lipid droplets,or if they are, their composition and thus reflective prop-erties differ from those of the ‘‘standard’’ lipid droplets.Another drawback of CRM - and of many contrastenhancing in vivo microscopic techniques—is that it isnot possible to label components of interests selectivelyfor reflection like in fluorescence microscopy.

Nevertheless, CRM is a valuable technique tostudy several aspects of the living material (see theSupp. Movies), including the motion of lipid droplets.As it is shown here, CRM offers a sensitive and reliableway to track lipid droplet motion (>99% sensitivity andover 97.5% selectibility) with high spatiotemporal reso-lution. The analytical methods used in the present workoffer �7–9 nm spatial resolution of centroid coordinatedetermination and �65 ms temporal resolution compara-ble with previous works (�2 nm spatial and 33 mstemporal resolution by Welte et al. [1998]). Both of theresolution values are at the technical maximum that mostof the confocal microscopes can achieve: during the fast-est bi-directional scanning, the alignment of the odd(outward) and even (homeward) lines are not as good ason a two-dimensional sensor array (such as a CCD).However, the use of a Nipkow-disc microscope and aCCD-detector can circumvent both technical limits.

The Drosophila Oocyte as an In Vivo ModelSystem of Motor-Mediated Transport

The lipid droplets have been the objects of MT-based motor activity studies in the developing Drosoph-ila embryo [Welte et al., 1998; Gross et al., 2000]. Wedescribe here the lipid droplets in the developing Dro-sophila egg primordia utilize MTs and the associatedmechanoenzymes to get transported. A large fraction(�90%) of the droplets travels along with the streamingooplasm. Although ooplasmic streaming seems to be asmooth flow of cytoplasmic components during low tem-poral resolution imaging, we demonstrate here that it is asaltatory motion of short runs followed by frequentpauses. Thus, the average short term velocity of the lipiddroplets is significantly higher (�450 nm/s) than theaverage long term velocity of either the yolk granules(�370 nm/s) or the lipid droplets (�320 nm/s). Still, theaverage long term speed of ooplasmic streamingreported here greatly exceeds the speed determined pre-viously (120 6 6.4 nm/s) [Serbus et al., 2005]. In thepresent work small field of views (20.91 3 20.91 lm2)were analyzed in which all the yolk granules and themajority of lipid droplets were streaming. However, the

yolk granules adjacent to the cortical membrane do notstream (apparent on images representing larger sectionsor the entire stage 10B-12 oocytes, see Supp. Movie 1).Some of those are accidentally dragged along with theooplasmic streams. Serbus et al. [2005] selected yolkgranules randomly for velocity measurements andincluded, most likely, some of the non-streaming yolkgranules. To our opinion these particles are not reallypart of the streaming ooplasm; they are rather proof ofthe supposition, that at least a fraction of the cytoplasmiccomponents travel passively during the streaming andthe centrifugal force exerted by the swirling ooplasmmay push some of them toward the cortex.

However, a subpopulation of the lipid dropletsescapes the drag of streaming. Their movements followthe classical motor-mediated transport: the high speeddiscrete runs are followed by either pauses or trackchanges, i.e. a change in the direction of motion. Desta-bilization of the MTs—by colchicine treatment or byE82K-a4-tubulin—results in the termination of the lin-ear motion and shows that fast displacement of the lipiddroplets depends on the presence of the MTs. Since thespeed of the moving particles (908 6 15 nm/s on aver-age) greatly exceeds the growing and shrinking rate ofthe dynamic MT ends (<50 nm/s) in the early stage 12egg primordia, it is very unlikely that the dynamic insta-bility of the MTs alone can drive lipid droplet motion.The remarkably great speed implies an involvement ofthe MT-based motor molecules, most likely the classicalKinesin-I and the cytoplasmic dynein for the plus- andthe minus-end directed runs, respectively. Interestingly,the speed measured for both the plus (848 6 17 nm/s)and the minus end directed motions (975 6 25 nm/s) isas high as determined for the conventional kinesin (600–1800 nm/sec) [Vale et al., 1985; von Massow et al.,1989; Bohm et al., 1999] and cytoplasmic dynein (800–1250 nm/s) [Paschal et al., 1987; Toba et al., 2006]in vitro, and about twice as high as measured in earlysyncytial embryos during phase I (407 6 49 nm/s and475 6 42 nm/s for plus and minus end, respectively)[Welte et al., 1998]. This suggests that the MT-basedtransport machinery during the stages of the fast ooplas-mic streaming is, not surprisingly, in a fully activatedstate. Consistent with this hypothesis, the physiologicalstoppage of the streaming—the deactivation of the trans-port elements—during stage 12 brings in a gradual slowdown in the non-streaming lipid droplet motion. How-ever, the cold recovery experiments clearly demonstratedthat the fast moving subpopulation of the lipid dropletsexists independently from the ooplasmic streaming andthe flowing cytoplasm has no significant influence ontheir motor-mediated motion.

The developing egg primordia appear ideal tools tostudy MT-based transport. They are easy to handle, have


been well characterized and are readily amenable tophysical, chemical and genetic manipulations. There aremutations that alter the MT-based transport during eggmaturation and disrupt or block embryogenesis whileallowing seemingly normal development of the egg pri-mordia [Belecz et al., 2001; Venkei and Szabad, 2005;Venkei et al., 2006]. While the Drosophila embryos areprotected by the impenetrable vitelline membrane, thismembrane is still permeable in the developing egg formost of the small molecules that may modulate MT in-tegrity and MT-based transport [Mahowald et al., 1983].Drug treatment opens an additional way to study realtime motor processivity and coordination. Finally, duringthe streaming stages (especially during early stage 12)the MT transport machinery is in a fully activated state.Thus, even the slightest alteration in any of the transportelements may result in perceptible and significantchanges of the lipid droplet transport. In summary, thedeveloping egg primordia in combination with the CRMtechnique provide a versatile system to study and under-stand vesicle motion and the underlying control mecha-nisms in vivo.

ACKNOWLEDGMENTS

Authors of this article express their gratitude totwo of the reviewers for their helpful comments on thefirst version of the manuscript.

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in vivo analysis of mt-based vesicle transport by confocal reflection microscopy

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