The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.


Introduction
Leishmania sp are flagellated trypanosomatid parasites responsible for widespread diseases in tropical and subtropical countries (http:// www.who.int/tdr/diseases/leish/default.htm). The parasite alternates between a flagellated extracellular form in the insect guts and an aflagellated intracellular form living in the parasitophorous vacuoles of mammalian macrophages. Such particularities and the evolutionary distance make it likely that there are sufficient differences in the biological pathways between parasites and hosts to find new parasite-specific drug targets. This is seriously needed due to the limited choice of available treatments, which are old, and the spreading of drug resistance. Basic research, accompanied by the recent publication of the complete genome sequence of several trypanosomatid species, including Leishmania [1,2], may help to find new approaches to control these diseases.
Intracellular traffic is an essential process in all living organisms. In humans, a number of severe diseases are caused by trafficking deficiencies [3]. It is not absurd to expect to find within such complex machinery a parasite-specific step that is exploitable to impair parasite traffic and viability. To our knowledge, such an achievement has not yet been reached, and it is much too early to know whether and when therapeutic applications may arise. However, there are already parasite-specific pathways candidates, for example the now well studied trafficking of GPI-anchored proteins in trypanosomes [4].
Vesicles represent the main tool of intracellular traffic and a great number of proteins are involved in their assembly, mobility and disassembly, including notably small G proteins, like members of the ARF/ARL (ADP-Ribosylation Factor/ADP-Ribosylation factor-Like) family [5][6][7][8]. There are 5 or 6 different ARFs and about a dozen ARLs among this family, depending on the species. The most studied are ARF-1 and ARF-6 with orthologues in yeast, plants, mammals [9], and even protozoa like Leishmania [10] and Trypanosoma [11,12]. ARL-1 has recently received much attention and orthologues have been studied in several species. ARL-1 has been localised to the trans-Golgi network (TGN) in yeast, mammals [13,14] and T. brucei [15]. Essential in Drosophila [16], ARL-1 maintains the integrity of the Golgi apparatus [15,17,18], and controls the vesicle traffic and the vacuole formation in yeast [19][20][21]. As for most other members of the family, myristoylation of its Nterminus is essential for its localisation and function [15,19,22,23].
Being a G protein, ARL-1 cycles between GDP-and GTPbound forms. In yeast, a nucleotide exchange factor, Ysl2p [24], and a GAP (GTPase-activating protein), Gcs1p [25], have been characterised; in mammals, only a yet uncharacterised ARF-GAP has been described [26]. Several effectors have been shown to interact with the GTP-bound form of ARL-1 [14,18,27], particularly the GRIP domain of several golgins/tethering factors. In the current model, such interactions allow the recruitment of these tethering factors to TGN membranes, which are essential for the vesicular traffic between the Golgi apparatus and endosomes [28][29][30][31][32]. This is probably how ARL-1 participates to the Golgi apparatus maintenance and vesicular traffic.
Recently, remarkable studies with the orthologue TbARL-1 have been reported in Trypanosoma brucei. TbARL-1 is only expressed in the bloodstream forms, where it is associated with the Golgi apparatus; RNAi experiments showed that TbARL-1 is essential for viability, Golgi apparatus maintenance and exocytosis in bloodstream forms, but has no effect in insect forms [15]. As Leishmania, this organism belongs to the Trypanosomatidae family. However, the two genus diverged possibly more than 100 million years ago [33] and they present many different features. To cite a few, the percentage of G/C in the T. brucei genome is lower than in L. major (41% versus 59.7% respectively) [34]; RNA interference (RNAi) is functional in T. brucei but impossible in Leishmania and concerning their life cycle, contrary to Leishmania, both T. brucei insect and mammalian (or bloodstream) forms are flagellated and extracellular, which has important physiological consequences.
We present here the characterisation of LdARL-1 in Leishmania. Contrary to T. brucei, LdARL-1 is expressed in both the insect and mammalian forms of the parasite. Design of a new mutant type, LdARL-1/T51N, corresponding to the GDP-bound blocked form, revealed that LdARL-1 cycles entirely within the TGN. This is contrary to previous data obtained in yeast and mammals with another mutant wrongly considered as the GDP-bound blocked form,. Expression of the dominant-negative mutant LdARL-1/ T34N had severe inhibitory effects on intracellular traffic, showing LdARL-1 involvement in the control of endocytosis, in intracellular trafficking from the TGN to the Lysosome/MVT (Multivesicular tubule) and the acidocalcisomes, and in the TGN targeting of the tethering factor pGRIP-2, a yet uncharacterised Leishmania GRIP domain-containing protein.

In vivo expression and subcellular localisation of ARL-1
A rabbit immune serum raised against the last 15 aa of LdARL-1 (underlined in Fig 1) allowed the detection of a unique 20 kDa band in promastigote and amastigote extracts of two strains of L. amazonensis, the BA125 strain (Fig. 2) and the LV79 strain (not shown). The expression of ARL-1 was similar in both forms of the parasite.
Untagged or GFP-fused LdARL-1 variants were expressed in L. amazonensis promastigotes using the pTEX [38] or the pNUS-GFPcH [39] vectors, respectively, both leaving the LdARL-1 Nterminus free. These vectors remain episomal and allow the expression of exogenous proteins. The abundance of the exogenous protein depends on the episome copy number, which can be manipulated within certain limits by the selective antibiotic concentration in the culture medium, and might also vary from cell to cell after unequal partitioning at mitosis. In this study, identical data were obtained with the transfectants expressing untagged or GFP-fused proteins.
Western blot analyses confirmed that the transfectants were expressing the proteins of interest (Fig 2). The endogenous 20 kDa LaARL-1 and a 48 kDa protein corresponding to LdARL-1-GFP were observed. Although LdARL-1 and LdARL-1-GFP were expressed at a higher level than the endogenous LaARL-1, this had no significant effect on the in vitro growth rate of the transgenic cell lines compared to parental cells ( Fig S2).
LdARL-1 GDP/GTP cycling G proteins cycle between GDP-and GTP-bound forms. Functional studies of these proteins have been done using mutants representing «GDP-bound» blocked (GDP to GTP exchange deficient) or «GTP-bound» blocked (GTPase-deficient) forms. The most recent characterisation of such mutants concerns human HsARF-6 [44]: the Q67L mutant represents the GTP-bound blocked form, T44N the GDP-bound blocked form, and the T27N, an empty form devoid of nucleotide, earlier erroneously considered as a GDP-bound form. The empty form of the native protein is a transient intermediate bound to the nucleotide-exchange factor; since the T27N mutant cannot bind GTP, the complex is  stable, the exchange process blocked and the cycling also when all the available nucleotide-exchange factor becomes complexed.
The expression of LdARL-1/T34N-GFP provided different observations. First, despite the comparable amount of endogenous LaARL-1 between the different transgenic cell lines, LdARL-1/ T34N-GFP expression level was much lower compared to the other variants (Fig 2). Accordingly, fluorescence microscopy observations revealed that, unlike the other mutants, only 15-20% of the cells were expressing detectable levels of LdARL-1/ T34N-GFP, suggesting that the protein was somehow toxic, and its expression repressed or counterselected. However, the growth rate of this cell line was comparable to the wild type cells ( Fig S2). Second, the fluorescence labelling obtained in the LdARL-1/ T34N-GFP-expressing cells was non-homogeneously distributed throughout the cytoplasm and totally excluded from the flagellum (Fig 5G), an observation easily discernable from the one made previously with the G2A mutant ( Fig 4B). This is consistent with the TGN disorganisation observed in mammals with the equivalent ARL-1/T31N mutant overexpression [18]. However, the unchanged localisation of LdARF-1-mRed ( Fig 5H) suggests differences in TGN targeting/organisation. Finally, localization of the double-mutant LdARL-1/G2AT34N (not shown) was undistinguishable from the G2A mutant, confirming again the necessity of N-terminal myristoylation.
In summary, LdARL-1 entire GDP/GTP cycling occurs within the TGN; the empty form is cytoplasmic.

LdARL-1 and intracellular trafficking
The various LdARL-1 mutants were used to further explore LdARL-1 function. As the TGN is a complex structure specialised in the sorting of molecules to the plasma membrane, the lysosome and other organelles, the putative involvement of LdARL-1 in intracellular traffic was investigated.
a/Exocytosis and endocytosis. The trypanosomatids are polarised cells in which endo-and exocytosis occur solely in a peculiar structure, the flagellar pocket. Upon exogenous expression of LdARL-1/T34N-GFP (empty), the flagellar pocket of the transgenic L. amazonensis cells appeared inflated, forming a gap at the anterior part of the cells (Fig 5G, fp); this was better resolved by labelling the pellicular and pocket membranes with Texas-Red-Concanavalin A, as seen for the cell pictured on Fig 6D-G and comparing to a cell expressing native LdARL-1-GFP (Fig 6A) or LdARL-1/T51N-GFP (GDP) (Fig 6B-C). This flagellar pocket inflation was similar to the observation made when endocytosis was inhibited by RNAi ablation of clathrin heavy chain in T. brucei [45], but could also result from enhanced exocytosis.
The activity of the secreted acid phosphatase (SAP) was determined in the culture medium of untransformed cells and expressing native or mutated LdARL-1-GFP; no significant difference was found (Fig S2), suggesting that SAP exocytosis was unmodified. Endocytosis was assessed by FM4-64 pulse-chase experiments (Fig 7). In Leishmania, this dye is readily internalised and targeted through endosomes to the final digestive compartment termed Lysosome/Multivesicular Tubule (MVT) [46]. In cells expressing LdARL-1-GFP (Fig 7A-D), LdARL-1/Q74L-GFP (not shown) or LdARL-1/T51N-GFP (not shown), FM4-64 endocytosis occurred as expected, with a progressive and massive FM4-64 labelling from the flagellar pocket to the MVT from 0 to 120 minutes. On the contrary, cells expressing LdARL-1/T34N-GFP showed endocytosis defect (Fig 7E-H); the FM4-64 uptake was blocked, the labelling, remaining in the area of the flagellar pocket up to 60 minutes (Fig 7E-G, arrows), was only seen progressing to the MVT at later times (120 min) and in much smaller amounts (Fig 7H, arrow); as comparison, the cell a in Fig 7F, which expresses LdARL-1/T34N-GFP, and the cell b which does not stresses the difference in FM4-64 internalization. This endocytosis defect was not due to clathrin depletion, as revealed by immunofluorescence with an anti-TbClathrin Heavy Chain immune serum (not shown).
c/Traffic to the acidocalcisomes. In trypanosomatids, acidocalcisomes are essential acidic organelles, probably lysosome-related [49]. They contain polyphosphates and calcium. Their acidic pH is maintained via the action of a membrane-bound pump, the proton pyrophosphatase (VP1). In T. brucei, TbVP1 is essential for the survival of the mammalian forms of the parasite [50].

LdARL-1 and GRIP domain proteins
In mammals and yeast, ARL-1/GTP has been shown to bind to the GRIP domain of large coiled-coil proteins, a conserved Cterminal sequence of about 45 residues sufficient to direct these proteins to the TGN [51,52]. These proteins are vesicle tethering factors [32]. In the Trypanosomatid genomes, only two proteins are predicted to possess GRIP domains. We called them pGRIP-1 (L.  mexicana [53]) and for the first time the full-length LdpGRIP-2. The three ORFs were fused with mRed at their N-terminus, leaving the GRIP domain at the C-terminus, and co-expressed in L. amazonensis together with LdARL-1-GFP or its mutants. As the results obtained were similar, only the data obtained with mRed-LdpGRIP-2 will be described.

The LdARL-1 gene in Leishmania and other Trypanosomatids
We identified and characterised a trypanosomatid member of the ARF/ARL family, the Leishmania LdARL-1. The gene was named ARL-1 after the highest percentage of identity with the corresponding mammalian and yeast homologues. It is conserved in other Leishmania (L. infantum, 100% identity; L. major, 3 amino acids are different; L. amazonensis, 4 amino acids are different; L. brasiliensis, 12 amino acids are different, one is added) and trypanosome species (T. brucei, 47 amino acids are different, 3 are added) whose sequence is available.
The situation in T. cruzi might be different as two sequences were reported. The first TcARL-1 has a comparable size to other species (45 different amino acids, 3 added). The second TcARL-1 has a 101 amino acid N-terminal extension; its ARL-1 domain has 3 different amino acids when compared to the first TcARL-1; 46 different amino acids, 3 added when compared to LdARL-1. However, the N-terminal extension is created by the deletion of a T in a stretch of 7 in the vicinity of the 59 side of the ARL-1 part. There is no obvious polypyrimidine tract upstream of the ATG and the predicted extended TcARL-1 protein cannot be Nmyristoylated because of the absence of a Glycine in position 2. Although there is an example in mammals of a chimeric protein with a C-terminal ARF-1 part and a N-terminal extension playing the role of an internal GAP (GTPase Activating protein) [54], the reality of the T. cruzi variant should be investigated in more detail to rule out the possibility of sequencing errors and/or to relate it with the existence of two divergent lineages of T. cruzi [55], eventually compatible with a 3 amino acid difference between the two TcARL-1 regions.
Conservation of the general organisation of the chromosomal region ( Fig S1) (even for the extended variant gene of T. cruzi) and the comparable percentages of identity between the different neighbouring orthologues across species suggest that all ARL-1s are functional homologues and that the TbARL-1 gene [15], erroneously annotated as TbARF-3 (GeneDB http://www.genedb.org/) should be renamed TbARL-1, unless functional data contradict this assumption.

N-myristoylation of LdARL-1
As for other members of the ARF/ARL family, the glycine in position 2 represents a myristoylation site; myristoylation is impossible for the LdARL-1/G2A-GFP mutated protein which remained diffuse within the cytoplasm, including the flagellum. Still, the mRed-LdpGRIP-2, mRed-LdGRIP or mRedTbGRIP localised to the TGN, showing that the LdARL-1/G2A-GFP was unable to compete with the endogenous native LaARL-1 and inhibit its function. Emphasising the essential character of myristoylation, the double-mutants LdARL-1/G2AQ74L-GFP (unMyr-GTP) and LdARL-1/G2AT34N-GFP (unMyr-empty) remained cytoplasmic, and their expression had no effect on the GRIP domain protein targeting. The absence of N-terminal myristate probably generates functionally inert LdARL-1 proteins, unable to bind to membranes and be recognised by a putative membrane bound targeting receptor/mechanism as suggested for HsARF-6 [56].

The LdARL-1 mutants and GDP/GTP cycling
Based on ras protein properties, mutations of conserved amino acids have been created to mimick GDP-bound or GTP-bound forms of the proteins of this superfamily. Thus, the last Threonine of the first motif of the GTP binding site (Fig 1) has been considered essential for GTP binding, its mutation leading to a drastic decrease in GTP affinity, hence producing a GDP-bound blocked form. However, due to the difficulties of determining the affinities for nucleotides, especially when they are low and if the proteins are insoluble, these affinities have been rarely looked over. After the crystal structure of several GDP-or GTP-bound ARF/ ARLs has been solved, the validity of the prediction for all the ras family proteins became questionable. In a recent detailed study of human ARF-6 [44], it was clearly shown that the mutant ARF-6/ T27N, considered as a GDP-bound blocked form, was in reality an empty form devoid of nucleotide since the Threonine 27 interacted with both GDP and GTP. Accordingly, in our work, the mutant LdLdARL-1/T34N was considered as an empty form, and the new mutant LdARL-1/T51N, a GDP-blocked form (cf Fig S3).
When expressed in Leishmania promastigotes, LdARL-1/T34N-GFP was delocalised to the cytoplasm, similarly to the equivalent mutants in mammalian cells [18]. However, LdARF-1-mRed localisation did not change, so that the TGN was not disorganised to the same extent as in mammals [18]. accordingly, it has been shown in mammals that Brefeldin A-induced disorganisation of the Golgi, which includes ARF-1 delocalisation to the cytoplasm, occurs within minutes of drug exposure, while it takes much longer for ARL-1 [17]. We found no effect of Brefeldin A in Leishmania (unpublished data), possibly because it was not internalised. Conversely, LdARL-1/T51N-GFP (GDP) localised to the TGN, and co-localised with LdARF-1-mRed. It was the same for LdARL-1/Q74L-GFP (GTP), which was different from mammals, where ARL-1/Q71L (GTP) led to an expansion of the Golgi and a delocalisation of ARF-1 [18]. It then appears that, in Leishmania, the entire LdARL-1 GDP/GTP cycling occurs within the TGN and the delocalisation to the cytoplasm of the «empty form» is a non-physiological event due to the expression of a transient form normally associated to the nucleotide exchange factor; the blocked empty form might act as a sink for the nucleotide exchange factor, thus forming a stable and inactive complex, as has been suggested for the equivalent mutant ARF-1/T31N [57]. From our data, one can predict that the unknown exchange factor is limiting, since the mutant LdARL-1/T34N-GFP is dominant-negative, i.e. blocks the endogenous native LaARL-1 function. The lack of physiological effect of LdARL-1/T51N-GFP suggests that this form does not stably associate with the exchange factor, the interaction being stabilised by the release of GDP. On the contrary, the unknown GAP is either non-limiting or not blocked by LdARL-1/Q74L-GFP since expression of this mutant does not block the endogenous native ARL-1 function. There is no obvious Ysl2p homologue in the Leishmania genome, the suggested yeast Arl-1p nucleotide exchange factor [24], nor Gcs1p, the suggested Arl-1p GTPase-activating protein [25]; their identification might help to better understand LdARL-1 GDP/GTP cycling.

LdARL-1 role in intracellular traffic as revealed by the dominant-negative mutant LdARL-1/T34N
Expression of LdARL-1/T34N-GFP significantly perturbed Leishmania intracellular trafficking. Endocytosis was completely blocked, trafficking of DPMS from the Golgi to the Lysosome/ MVT, and of the membrane-bound vacuolar proton pyrophosphatase to the acidocalcisomes, was interrupted. The fate of these markers remains enigmatic; they were possibly degraded or misdirected and exocytosed, which would be reminiscent of ScArl-1 KO, where lucifer yellow uptake was reduced and the vacuolar Carboxypeptidase Y secreted instead of being vacuolar [21]. In Leishmania, exocytosis seemed unaffected; however, only the SAP marker (secreted acid phosphatase) could be investigated and changes changes in exocytosis might not have been detected since only 15-20% of the cells expressed the mutated protein.
The acidocalcisomal defect observed with LdARL-1/T34N-GFP expression resembled the vacuolar defect obtained in yeast with the mutant Arl-1p/D151G mutant, which proved also dominant-negative [20], but the mechanism of action is probably different, because, from the protein structures, the D151 of Arl-1p does not interact with GDP or GTP. At least some of the membrane-bound proteins of acidocalcisomes originate from the TGN and a functional LdARL-1 protein is essential for their transport to their final compartment. Whether LdARL-1-mediated targeting to acidocalcisomes is direct from the TGN or necessitates endosomal/lysosomal intermediates remains unknown [49] and might be investigated in the future if/when appropriate Leishmania protein markers are identified.

ARL-1-dependent Golgi targeting of GRIP domain containing Golgins
The GRIP domain is a C-terminal 45 aa long domain present in at least five human and one yeast proteins [32,60]. Essential for the TGN targeting of these proteins, it is conserved in trypanosomatids and in Leishmania, a T. brucei GRIP domain is sufficient to target GFP to the TGN [53].
At least for Leishmania, the model of ARL-1/GRIP domain interaction and Golgi recruitment might be slightly modified. GRIP domain proteins and ARL-1 under both GDP and GTP forms are localised to the TGN. Both LdARL-1 forms are probably associated to membranes as HsARF-6 [56]. The GTP form may recruit GRIP-domain proteins from the Golgi soluble compartment to Golgi membranes and the GDP form releases them. This is consistent with the fact that in Brefeldin A-treated mammalian cells, ARL-1 is delocalised from the Golgi membranes while GRIP domain proteins are not [27], showing that, at least in mammals, ARL-1 is not necessary for the maintenance of GRIP domain proteins in the TGN. In the case of Arl1p knockout in yeast, the GRIP domain protein Imh1p was mislocalised [29] possibly because it could not reach the Golgi at all.
The recruitment of GRIP domain proteins to the Golgi also requires the presence of another small G protein, Scarl3p, and an integral membrane protein, ScSys1p [61,62]; Scarl3p/GTP and / GDP forms were included in the study, but the mutant used for the GDP form might also be an «empty form»; the model should be revisited according to this new interpretation.
Concerning Leishmania, we have found a putative Scarl-3p homologue (not yet functionnaly investigated), but there is no obvious candidate for ScSys1p. Much work remains to be done for the understanding of this aspect of the Golgi structure/function.

Significance of LdARL-1 in the biology of Leishmania cells
The LdARL-1 gene is expressed in both promastigotes (insect forms) and amastigotes (mammalian forms) of L. donovani and L. amazonensis. This differs from T. brucei, where TbARL-1 is expressed only in the bloodstream forms of the parasite (i.e. the mammalian forms) and is essential for their viability, as revealed by RNAi experiments [15]. Although it has not been formally demonstrated and in spite of being annotated as ARF-3 in the T. brucei database (Tb927.7.6230, GeneDB http://www.genedb.org/), this TbARL-1 is very likely the functional homologue/orthologue of LdARL-1 given the synteny of the homologous chromosomal region of several trypanosomatids (Fig S1). The differential expression seen in T . brucei forms probably reflects differences in the physiology of these cells: endocytosis is minimal in T. brucei insect forms, while it is extraordinarily active in bloodstream forms [4], which correlates well with LdARL-1 involvement in endocytosis. In Leishmania, LdARL-1 and endocytosis must be active in all forms of the parasite.
Finally, the fact that LdARL-1 could not complement the yeast arl-1D deletion mutant [21] (not shown) reveals that, even for evolutionary conserved pathways such as intracellular traffic, interspecies differences exist, which might hopefully be exploited against deleterious (from the human point of view only) organisms in the future.
Protein extraction and western blotting were done as previously described [58] except for the revelation, done with a rabbit anti IgG peroxidase conjugate (1:10000 dilution) and an ECL revelation kit (Amersham). The secreted acid phosphatase (SAP) activity was determined as previously [58] according to [67]. Briefly, after seeding the cells at a density of 2.5 10 6 per ml, aliquots of the culture medium were removed every following day for 5 days, filtered through a 0.22 mm pores membrane to remove cells and debris; 138 ml supernatant were then incubated in a final volume of 200 ml with 50 mM sodium acetate pH 4, 0.1% bmercaptoethanol (v/v), 50 mM para-nitrophenyl phosphate for 30 min at 37uC; the reaction was stopped with 800 ml sodium hydroxide 2 M and the absorbance of the released paranitrophenolate at 410 nm determined; the amount of paranitrophenolate ion was calculated considering an extinction coefficient of 17.8 mM 21 , and the data presented as nmoles para-nitrophenyl phosphate hydrolyzed per minute and milliliter of supernatant.
For the flagellar pocket and plasma membrane visualisation, cells were washed twice with RPMI-1640 medium plus 1% goat serum and incubated in RPMI-1640 plus 50 mg/ml biotin-labelled Concanavalin A (Sigma) for 30 min at 24uC. After two PBS washes, cells were fixed for 1 hour at room temperature with 2% paraformaldehyde and further incubated for 1 hour with 10 mg/ ml Texas-Red-Streptavidine conjugate (Molecular Probes).
For acidocalcisomal pyrophosphate staining [50], cells were washed twice in 116 mM NaCl, 5 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM K-Hepes, pH 7.4, and incubated for 10 min at 30uC in PBS with 10 mg/ml DAPI. Cells were spread on coverslips by centrifugation and observed alive quickly thereafter.
Observations were done with an Axioplan 2 Zeiss fluorescence microscope and a 1006 oil lens. Images were acquired with a Princeton Instruments camera and analysed with Metaview TM (Universal Imaging).