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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01344

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lis-1 is required for dynein-dependent cell division processes in C. elegans embryos

Swiss Institute for Experimental Cancer Research (ISREC), Ch. des Boveresses 155, 1066 Epalinges/Lausanne, Switzerland

View larger version (120K): [in a new window]   Fig. 5. GFP–LIS-1 localizes to kinetochores independently of dynein. (A-C,E-G) Spinning-disc confocal time-lapse fluorescence microscopy of one-cell-stage embryos derived from homozygous lis-1(1550) adults expressing GFP–LIS-1 with no further treatment (A), after icp-1(RNAi) (B), hcp-4(RNAi) (C), dhc-1(RNAi) (E), dnc-1(RNAi) (F) or dnc-2(RNAi) (G). A single image during late prometaphase or equivalent stage is shown here; see also corresponding Movies 9, 13, 14, 17, 18 and 19 (in supplementary material, http://jcs.biologists.org/cgi/content/full/117/19/4571/DC1). Insets show 2.5-fold digitally magnified views of the region encompassing chromosomes. Note that enrichment in the vicinity of chromosomes during prometaphase is absent in hcp-4(RNAi) embryos (C), but is present in all other cases. Six to twelve embryos were imaged for each genotype, with a similar outcome. (D) Images from confocal time-lapse fluorescence microscopy of dhc-1(RNAi) one-cell-stage embryo expressing GFP-TUB and GFP-HIS (see also corresponding Movie 16, in supplementary material). Time elapsed is shown in minutes and seconds. Arrows indicate position of chromosomes derived from the male pronucleus. Arrowheads point to the single centrosome visible in this focal plane.

View larger version (35K): [in a new window]   Fig. 1. Characterization of LIS-1 antibodies. (A) Western blots of protein extracts from wild-type, lis-1(RNAi) and lis-1(t1698) embryos, probed with LIS-1 antibodies and reprobed with -tubulin (TUB) antibodies. We detected a 15 kDa band in some extracts from mixed developmental stages of homozygote lis-1(t1698) mutant animals (not shown). (B) Wild-type, lis-1(RNAi) and lis-1(t1550) embryos stained with LIS-1 and -tubulin antibodies. Wild-type embryos fixed and stained on the same slides as lis-1(RNAi) or lis-1(t1550) embryos are shown. Bar, 10 µm. (C) Quantification of average LIS-1 reactivity, expressed as a fraction of wild-type values for wild-type (100%, n=18), lis-1(RNAi) (5.2%; n=9) and lis-1(t1550) or lis-1(t1698) [referred to collectively as lis-1(mutant)] (2.9%; n=11) embryos.

View larger version (106K): [in a new window]   Fig. 2. lis-1 is required for pronuclear migration, centrosome separation and bipolar spindle assembly. (A-C) Images from time-lapse DIC microscopy of wild-type (A), lis-1(t1550) (B) and lis-1(RNAi) (C) one-cell-stage embryos (see also corresponding Movies 1-3, in supplementary material, http://jcs.biologists.org/cgi/content/full/117/19/4571/DC1). In this and other figures, anterior is to the left, posterior to the right, bars represent 10 µm, and all panels of a kind are at approximately the same magnification, unless stated otherwise. Time elapsed is shown in minutes and seconds, with 00:00 corresponding to the time of maximal pseudocleavage furrow ingression. Arrowheads indicate the position of centrosomes as inferred primarily from fluorescence imaging of equivalent embryos expressing GFP-TUB. Arrows indicate anterior-most boundary of a region depleted of yolk granules that emanates from the centrosomes and extends towards the anterior following breakdown of the male pronuclear envelope. Note that breakdown of the male pronucleus precedes that of the female pronucleus as is generally the case when the two pronuclei are not juxtaposed (Gönczy et al., 1999a). Note also that furrowing initiates but does not go to completion towards the cell anterior in lis-1(t1550) and lis-1(RNAi) embryos, presumably reflecting a local minimum of microtubule density (Dechant and Glotzer, 2003). (D-G) Wild-type one-cell-stage embryo during metaphase (D) and lis-1(t1550) embryo at equivalent stage (E), as well as wild-type four-cell-stage embryo (F) and lis-1(t1550) embryo at equivalent stage (G) stained with antibodies against ZYG-9 (red) and -tubulin (green) and counterstained with Hoechst to view DNA (blue). Yellow spots are indicative of ZYG-9 staining at centrosomes (arrowheads). Arrow indicates anterior-most boundary of microtubules emanating from the centrosomes and abutting chromosomes from the male gamete [see also Movie 16 (supplementary material) for similar feature in dhc-1(RNAi) embryo]. Images are projections of two consecutive 1 µm confocal slices. (H) Mean centrosome-to-centrosome distance, measured in live embryos expressing GFP-TUB, in wild-type (n=7), lis-1(RNAi) (n=15) and dhc-1(RNAi) (n=12) embryos at the time of male pronuclear envelope breakdown. Bars represent s.d.

View larger version (104K): [in a new window]   Fig. 3. LIS-1 distribution in early embryos. (A-I) Wild-type embryos stained with antibodies against LIS-1 and -tubulin; upper panels show LIS-1 staining, lower panels the merge of LIS-1 (red), -tubulin (green), and Hoechst counterstain to view DNA (blue). Bar, 10 µm. (A-D) One-cell stage. (A) Early prophase; LIS-1 is excluded from pronuclei. (B) Pronuclear meeting; LIS-1 is no longer excluded from pronuclei. (C) Late prometaphase; arrow points to enriched LIS-1 in the vicinity of chromosomes, arrowheads to LIS-1 depletion at the aster centres. (D) Telophase; arrow indicates LIS-1 enrichment at the ingressing central cortex, arrowheads regions of LIS-1 enrichment in the vicinity of microtubule asters. Square 1 indicates an area of high microtubule density, square 2 an area of low microtubule density. Note that LIS-1 staining is more intense in square 1. (E) Two-cell stage; note enrichment of LIS-1 on the P1 spindle (arrowhead) and the cell cortex between AB and P1 (arrow). (F) Four-cell stage; arrowheads point to LIS-1 enrichment in ABa and ABp prophase nuclei. (G,H) Magnified views of the nucleus in the AB blastomere of a two-cell-stage embryo (G) and of the region containing chromosomes in a prometaphase one-cell-stage embryo (H). Arrows point to LIS-1 enrichment at the nuclear periphery (G) and in the vicinity of chromosomes (H). Bar, 2 µm. (I) Three-fold magnification of P1 spindle in panel E. Shown are immunostaining of LIS-1, -tubulin and the merge of LIS-1 (red), -tubulin (green) and DNA (blue). Arrow points to an apparent microtubule plus end that does not colocalize with a focus of LIS-1, arrowhead to one that does (see also corresponding Movie 8; supplementary material, http://jcs.biologists.org/cgi/content/full/117/19/4571/DC1). (J,K) GFP–LIS-1 transgenic two-cell-stage (J) and four-cell-stage (K) embryos stained with antibodies against GFP and -tubulin; upper panels show GFP staining, lower panels the merge of GFP (red), -tubulin (green), and Hoechst counterstain to view DNA (blue). Images are projections of up to three consecutive 1 µm confocal slices.

View larger version (72K): [in a new window]   Fig. 4. Dynamics of GFP–LIS-1 localization. (A,B) Spinning-disc confocal time-lapse fluorescence microscopy of one-cell-stage (A) and four-cell-stage (B) embryos derived from homozygous lis-1(1550) adults expressing GFP–LIS-1 (see also corresponding Movies 9-10, in supplementary material, http://jcs.biologists.org/cgi/content/full/117/19/4571/DC1). Time elapsed since the beginning of each movie is shown in minutes and seconds; live embryos in this and all other figures are 50 µm long. Note enrichment at nuclear periphery during prophase (A, time 01:55 and 03:50; B, time 00:00 and 02:15), entry in pronuclei/nuclei during late prophase (A, time 03:50; B, time 02:15), as well as further enrichment in the vicinity of chromosomes during prometaphase (A, time 05:40; not visible in B due to orientation of spindle in ABa and ABp). (C) Quantification of average pixel intensities in the nucleus and a comparable area in the cytoplasm of the four-cell-stage embryo displayed in panel B; results are shown for the time interval between the moment GFP–LIS-1 enrichment becomes apparent at the nuclear periphery and NEBD. Similar plots were obtained in three other embryos. (D,E) FRAP experiments. Images from representative confocal time-lapse sequences of four-cell-stage embryos derived from homozygous lis-1(1550) adults expressing GFP–LIS-1 (D) or wild-type animals injected with FITC-labelled Dextran 70 kDa (D) (see also corresponding Movies 11 and 12, in supplementary material). Time elapsed is shown in minutes and seconds. Photobleaching of a 4.5 µm2 square (indicated in white) started at time –00:02 and ended at time 00:00. Note very rapid recovery (t1/2 10 seconds) of fluorescence for both embryos.

View larger version (102K): [in a new window]   Fig. 6. LIS-1 targeting to the cell cortex, the nuclear periphery and kinetochores is microtubule-independent. Wild-type (C) or lis-1(1550) embryos rescued by GFP–LIS-1 expression (A,B,D,E) treated with tba-2 (RNAi) and stained with antibodies against LIS-1 and -tubulin; upper panels show LIS-1 staining, lower panels the merge of LIS-1 (red), -tubulin (green), and Hoechst counterstain to view DNA (blue). (A) One-cell-stage embryo prior to pronuclear envelope breakdown; note that GFP–LIS-1 is excluded from pronuclei. (B) One-cell-stage embryo during mitosis; note intense GFP–LIS-1 in the vicinity of condensed chromosomes (arrow). (C) High magnification view of chromosomes in an embryo at a later cell cycle. Embryo posterior is up; bar, 2 µm. Note that LIS-1 enrichment follows the position of chromosomes. (D) Embryo at a later cell cycle; note cortical enrichment of GFP–LIS-1 (arrow). (E) Later-stage embryo during prophase; note enrichment at the nuclear periphery (arrow). Images are projections of up to three consecutive 1 µm confocal slices.

View larger version (71K): [in a new window]   Fig. 7. Relationship between lis-1 and dynein/dynactin. (A,B) Embryo at the two-to-four cell stage transition expressing GFP–LIS-1 and stained with antibodies against DHC-1 and GFP; upper panels shows DHC-1, middle panel shows GFP, lower panel the merge of GFP (red), DHC-1 (green), and Hoechst counterstain to view DNA (blue). Panel A (approximately 6x10 µm) shows high magnification of the P1 nucleus during late prophase, panel B (approximately 3x5 µm) a cytoplasmic area from the AB cell in the same embryo. Bottom of diamond points to a focus of GFP–LIS-1 that does not coincide with intense DHC-1 staining, arrow to a focus of DHC-1 that does not coincide with intense GFP–LIS-1 staining, arrowhead to a focus of coincident DHC-1 and GFP–LIS-1 staining. (C,D) Wild-type (C) and lis-1(t1550) (D) two-cell-stage-equivalent embryos stained with antibodies against DHC-1 and -tubulin. Upper panels show DHC-1 staining, lower panels the merge of DHC-1(red), -tubulin (green) and Hoechst counterstain to view DNA (blue). Note that DHC-1 enrichment at the nuclear periphery, in the vicinity of microtubule asters (arrowheads) and at the cell cortex (arrows) are all somewhat diminished in lis-1(t1550) mutant embryos compared with wild-type. In rare lis-1(t1550) embryos, enrichment of DHC-1 at specific subcellular locations appeared to be absent (not shown). Bar, 10 µm. (E-G) dhc-1(RNAi) (E), dnc-1(RNAi) (F) or dnc-2(RNAi) (G) embryos at the second cell cycle stained with antibodies against LIS-1 and -tubulin. Upper panels show anti-LIS-1 staining, lower panel the merge of LIS-1(red), -tubulin (green) and Hoechst counterstain to view DNA (blue). Note that LIS-1 is still present at the cell cortex (arrows), albeit to somewhat lower levels than in the wild-type (compare with Fig. 3D). Note also that LIS-1 enrichment along chromosomes is visible in the dnc-2(RNAi) embryo (G, arrowhead). Moreover, note that LIS-1 becomes trapped in nuclear compartments after the first failed cell division in this particular dhc-1(RNAi) embryo (E); this was not observed in all dhc-1(RNAi) embryos examined, and only rarely after inactivation of dynactin components.

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