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First published online 1 September 2005
doi: 10.1242/jcs.02530

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Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis

Yannick Gachet^* and Jeremy S. Hyams^,

Department of Biology, University College London, Gower Street, London, WC1E 6BT, UK

View larger version (53K): [in a new window]   Fig. 1. Endocytosis in fission yeast is coincident with regions of cell growth and cytokinesis. (A) Time course of FM4-64 uptake in living cells at different stages of the cell cycle. Left-hand column, pre-NETO cell. Middle column, post-NETO cell. Right-hand column, dividing cell. Each cell is initially shown as a phase-contrast image (Ph). Cells are oriented with the old end to the right. In the pre-NETO cell fluorescence is initially associated with the old end only but begins to appear also at the new end as the cell passes NETO (50 minutes). In the post-NETO cell both poles are initially fluorescent but the old end is brighter. In dividing cells, fluorescence accumulates at the cell equator. Arrows indicate the changes in growth polarity as individual cells progress through the cell cycle. (B) FM4-64 uptake is by endocytosis. FM4-64 fluorescence was followed in live cells at different temperatures and in the presence of sodium azide. Inhibition at low temperature and energy dependence are characteristics of endocytosis. (C) Cells treated with sodium azide prior to addition of FM4-64. In these conditions the dye binds to the entire cell surface. (D) FM4-64 uptake is by endocytosis. FM4-64 uptake is inhibited in ypt7. Ypt7 is a Rab GTPase that is essential for vesicle fusion. WT, wild type. (E) Localisation of endocytosis in tea2. In cells lacking the kinesin-related protein Tea2, cell growth is directed to the tip of the lateral branch, which is also the site of endocytosis. The nucleus is stained with Hoechst. (F) Endocytosis in nitrogen-starved, G1-arrested wild-type cells co-stained with Calcofluor to reveal the cell wall. Bright staining with Calcofluor identifies the old end as the single site of endocytosis. (G,H) Endocytosis switches from monopolar to bipolar at NETO. In cdc10-129 cells arrested in G1 by growth at 36°C, endocytosis is monopolar. Following shift to the permissive temperature (25°C), cells pass NETO and endocytosis becomes bipolar coincident with the appearance of actin at both cell poles. Bars, 10 µm (A,C,E,H); 4 µm (F).

View larger version (37K): [in a new window]   Fig. 2. Endocytosis in fission yeast passes from the cell membrane to the vacuole via intermediate prevacuolar compartments. (A) Vacuoles were prelabelled with CDCFDA and then exposed to FM4-64. After 15 minutes, FM4-64 labels prevacuolar compartments not stained with CDCFDA. (B) Quantification of the experiment in A. The number of CDCFDA-stained vacuoles (green line, V) remains constant throughout the experiment. The number of FM4-64-stained PVC initially rises but subsequently falls as fluorescence is transferred to the vacuolar membrane (red line, PVC). The number of FM4-64-labelled vacuoles (red line, V) increases with time. (C) The same experiment as in A but vacuoles were first fused by incubation in water for 60 minutes. (D) The number of CDCFDA-stained vacuoles (green line, V) remains constant through the experiment but the number of FM4-64-labelled PVC reaches a plateau as transfer to the vacuolar membrane is blocked (red line, PVC). Bar, 10 µm.

View larger version (60K): [in a new window]   Fig. 3. Internalised FM4-64 is rapidly transferred to vesicles containing Syb1. (A) Endocytosed FM4-64 (red) is rapidly transferred to Syb1 vesicles (green) as judged by the overlap of the two signals (yellow) after less than 1 minute. This series shows a pre-NETO cell. Note that Syb1 vesicles at the non-growing new end remain green. (B) Ratio of green and red fluorescence at the old (O) and new (N) ends pre and post NETO. In pre-NETO cells, FM4-64 uptake (red bars) is confined to the old end despite there being a bias to Syb1 vesicles (green bars) at the opposite pole. In post-NETO cells the two poles are equivalent. Error bars indicate s.e.m. Bar, 10 µm.

View larger version (46K): [in a new window]   Fig. 4. Endocytosis in fission yeast is associated with actin. (A) Crn1-GFP cells were labelled with FM4-64 examined under the microscope within 1-2 minutes. A subset of endocytic vesicles (red) and Crn1-GFP spots (actin patches, green) are superimposable (yellow). (B) Endocytosis is inhibited Lat B. Crn1-GFP cells were treated with 10 µM Lat B for 10 minutes prior to the addition of FM4-64. Fluorescence remained associated with the cell membrane, particularly at the cell equator, and with some residual structures at the cell membrane. No transfer to internal compartments was observed. (C,D) Effect of drugs on FM4-64 and CDCFDA uptake. Whereas FM4-64 uptake was inhibited by Lat B, CDCFDA uptake was not. TBZ had no effect on the uptake of either probe. (E) In cells preloaded with FM4-64 3 minutes prior to the addition of Lat B or TBZ (arrow) the dye trafficked normally to the vacuole (F). Thus later events of the endocytosis pathway are independent of both actin and microtubules. Bars, 10 µm.

View larger version (60K): [in a new window]   Fig. 5. Endocytosis in fission yeast is associated with actin. (A,B) FM4-64 uptake is inhibited in the actin mutant cps8 even at the permissive temperature of 25°C (B). Note the bright fluorescence at the cell membrane, particularly at the equator of dividing cells. (A,C) FM4-64 uptake is not inhibited in the -tubulin mutant nda2 at the restrictive temperature (36°C). (D) FM4-64 internalisation is inhibited in the Arp2/3 complex mutant arp2 at the restrictive temperature but not in the formin mutant for3 (E). (F,G) Syb1 vesicles at the poles and the equator show different sensitivity to latrunculin. Whereas vesicles in dividing cells collapse to the cell membrane in low latrunculin concentrations, higher concentrations are required to disrupt Syb1 vesicles at the poles of interphase cells. (G) Syb1-GFP collapses to the membrane in latrunculin-treated cells (Lat A). Bars, 10 µm.

View larger version (54K): [in a new window]   Fig. 6. Equatorial endocytosis in fission yeast is associated with actin. (A) A dividing Crn1-GFP cell treated with FM4-64. Transient colocalisation of endocytic vesicles and actin patches is observed at the equator as at the cell poles. (B) Mitosis in a atb2-gfp cell showing the accumulation of endocytic vesicles in the region between the separating sister chromatids and the disappearance of vesicles at the poles. As the sequence focuses on the elongation of the spindle, endocytic vesicles appear out of focus. (C) Endocytosis at cell division in atb2-gfp for3 showing the continuation of polar endocytosis in this mutant. (D) Polar endocytosis continues through cytokinesis in for3 mutants. (E) Polar endocytosis continues at cell division in atb2-gfp cdc12. (F) Equatorial endocytosis is misplaced in mid1 cells. Bars, 10 µm.

View larger version (33K): [in a new window]   Fig. 7. Relocation of endocytosis from the poles to the equator requires a functional SIN. (A,B) In cdc7, sid1 and sid2 cells the site of endocytosis moves to the equator at the permissive (25°C), but not the restrictive (36°C) temperatures. Myo2 ring formation is independent of SIN function and hence, endocytosis. For sid1 and sid2 cells the images shown are merged Myo2-GFP, FM4-64 and DAPI. (C) Quantification of equatorial endocytosis in wild-type cells (WT) and SIN mutants at 25°C (closed bars) and 36°C (open bars). Bar, 10 µm.

View larger version (35K): [in a new window]   Fig. 8. Equatorial endocytosis is dependent upon exocyst function. (A) Equatorial endocytosis was scored in dividing cells in the presence of BFA using ethanol (Eth) as a control and in sec8-1 cells at the permissive (25°C) and restrictive (36°C) temperatures. (B) Sec8-GFP vesicles and FM4-64-labelled vesicles are largely distinct although some exchange may occur between these compartments. Bar, 10 µm.

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