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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.007732

Journal of Cell Science 120, 2318-2327 (2007)
Published by The Company of Biologists 2007
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Research Article

Possible roles of the endocytic cycle in cell motility

David Traynor and Robert R. Kay*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

* Author for correspondence (e-mail: rrk{at}mrc-lmb.cam.ac.uk)

Accepted 9 May 2007


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Conclusion
 Materials and Methods
 References
 
Starving, highly motile Dictyostelium cells maintain an active endocytic cycle, taking up their surface about every 11 minutes. Cell motility depends on a functional NSF (N-ethylmaleimide sensitive factor) protein - also essential for endocytosis and membrane trafficking generally - and we, therefore, investigated possible ways in which the endocytic cycle might be required for cell movement. First, NSF, and presumably membrane trafficking, are not required for the initial polarization of the leading edge in a cyclic-AMP gradient. Second, we can detect no evidence for membrane flow from the leading edge, as photobleached or photoactivated marks in the plasma membrane move forward roughly in step with the leading edge, rather than backwards from it. Third, we find that the surface area of a cell - measured from confocal reconstructions - constantly fluctuates during movement as it projects pseudopodia and otherwise changes shape; increases of 20-30% can often occur over a few minutes. These fluctuations cannot be explained by reciprocal changes in filopodial surface area and they substantially exceed the 2-3% by which membranes can stretch. We propose that the endocytic cycle has a key function in motility by allowing adjustment of cell surface area to match changes in shape and that, without this function, movement is severely impaired.

Key words: Cell motility, Endocytosis, Membrane flow, Surface area, Dictyostelium, NSF, Photobleaching, Photoactivation


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Conclusion
 Materials and Methods
 References
 
Crawling cell movement and chemotaxis are conserved behaviours of many eukaryotic cells, with striking similarities apparent between such distant relatives as leukocytes and Dictyostelium amoebae (Devreotes and Zigmond, 1988Go; Parent, 2004Go). According to current ideas, chemotaxing cells process the external gradient of chemo-attractant to produce a sharply defined activated zone in their plasma membranes, which is marked by the local accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] (Parent et al., 1998Go), though chemotaxis itself does not depend on an PtdIns(3,4,5)P3 gradient (Hoeller and Kay, 2007Go; Loovers et al., 2006Go). Effector molecules are recruited to this zone, leading ultimately to the local polymerization of actin and expansion of a pseudopod (Mitchison and Cramer, 1996Go; Pollard and Borisy, 2003Go). It is notable that this scheme, though satisfying, does not require a major involvement of the plasma membrane in cell movement, beyond signal processing and providing adhesion sites to the substratum.

By contrast, genetic experiments in Dictyostelium have shown an unexplained dependence of cell motility on the membrane trafficking proteins clathrin and NSF (N-ethylmaleimide sensitive factor) (Wessels et al., 2000Go; Thompson and Bretscher, 2002Go), and several possible links between membrane trafficking and cell movement have been proposed, some dating back many years. These include the delivery of adhesion molecules to the front of the cell (Bretscher, 1984Go; Lawson and Maxfield, 1995Go), polarizing the plasma membrane (Kriebel et al., 2003Go; Thompson and Bretscher, 2002Go) and generating membrane flow. Membrane flow was first suggested by the rearward movement of particles attaching to the front of a migrating cell (Abercrombie et al., 1970Go) and subsequently proposed as an alternative motive force to drive the cell forward (Bretscher, 1984Go), though its existence in certain cells is strongly disputed (Sheetz et al., 1989Go; Lee et al., 1990Go).

One idea not so widely considered is the possibility that the endocytic cycle may be required by moving cells to allow them to adjust their surface area. Many moving cells can change shape radically as they move. For instance, Dictyostelium cells can expand their leading edge by myosin II-dependent blebbing (Yoshida and Inouye, 2001Go; Yoshida and Soldati, 2006Go; Langridge and Kay, 2006Go) and they alternate between rounded and elongated morphologies as they move (Wessels et al., 1998Go), with pseudopodia often projected from the front before the rear is withdrawn (Weber et al., 1995Go). Geometrical considerations suggest that these behaviours may produce transient increases in surface area, though this has not been quantified. It is crucial to know the magnitude of such changes, since membranes can only be stretched by 2-3% before rupturing (Mohandas and Evans, 1994Go) and changes beyond this would require some dedicated mechanism to supply additional surface area, such as an increase in exocytosis.

All of theses scenarios require moving Dictyostelium cells to maintain an active endocytic cycle yet, paradoxically, fluid phase endocytosis is almost quiescent in the starving and highly motile cells that are often studied (Kayman and Clarke, 1983Go; Maeda and Kawamoto, 1986Go). However, the more relevant membrane endocytosis has not been measured and it has been shown that cells grown on bacteria have a similarly low rate of fluid uptake as starving cells, yet take up membrane rapidly (Aguado-Velasco and Bretscher, 1999Go).

In this paper, we first show that membrane endocytosis remains active in starving cells and then investigate three possible ways in which the endocytic cycle might be involved in the movement of Dictyostelium cells: to polarize the leading edge; to produce membrane flow; or in surface area regulation.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Conclusion
 Materials and Methods
 References
 
Endocytosis by starving, aggregation-competent cells
Chemotaxis to cAMP is studied in cells that have been starved for a few hours, to allow them to initiate development and hence gain sensitivity to cAMP (`aggregation-competent' cells). We measured membrane endocytosis in such cells using the lipophilic dye FM1-43 (Betz et al., 1996Go; Aguado-Velasco and Bretscher, 1999Go). This only fluoresces when partitioned into a membrane, yet cannot cross this membrane and so only enters cells by endocytosis.

Within seconds of adding FM1-43 to a suspension of aggregation-competent cells, their plasma membranes become fully stained, and fluorescence then gradually accumulated in small cytoplasmic puncta, which were not fully resolved (Fig. 1A). Staining of the contractile vacuole, as reported with the related dye FM4-64 (Heuser et al., 1993Go), is not seen in short time courses with FM1-43. When the dye is washed out, surface labelling is lost, but internal staining remains, showing that dye has been internalized (not shown).


Figure 1
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  		Fig. 1. Endocytosis of membrane and fluid. Ax2 cells, grown in axenic medium, were washed free of growth medium and used immediately (vegetative cells) or starved for approximately 5 hours with periodic cAMP pulses after the first hour (aggregation competent cells). (A) Aggregation-competent cells stained with FM1-43: after a few seconds labelling is restricted to the cell surface, but by 100 seconds, there is additional punctate labelling within the cell, presumably as a result of endocytosed membrane (confocal sections). (B) Membrane endocytosis by vegetative ({square}) and aggregation-competent ({circ}) cells determined using FM1-43, with 10 mM azide added as indicted (Figure 1, Figure 1). Results are given as the fraction of the cell surface taken up by normalizing to the first measurement after mixing dye and cells. Inset shows a continuous trace made by adding FM1-43 directly to the cuvette illustrating the nearly instantaneous increase in fluorescence. (C) Fluid-phase endocytosis by vegetative ({square}) and aggregation-competent cells ({circ}) determined using FITC dextran. Values are mean ± s.d. of three experiments.

 
Endocytosis was quantified in cell suspensions by measuring the increase in fluorescence over time with a fluorimeter (Fig. 1B, Table 1). The almost instantaneous increase following addition of FM1-43 to cells was taken as a measure of plasma membrane surface area (Fig. 1B, inset), and subsequent changes expressed relative to this. Azide almost completely blocks uptake, showing that it is energy dependent, and it is also blocked in an NSF mutant (see later; Fig. 1B, Table 1). Uptake is linear for around 5 minutes and continues at a much slower rate for at least 1 hour, implying that the compartment responsible for the initial uptake rapidly saturates and after this time dye is released by exocytosis at a rate almost equal to its uptake. From the initial rate of dye uptake, we calculate that starving cells take up an area equivalent to their entire plasma membrane every 10.8 minutes, which is very similar to the rate for vegetative cells (Table 1) (Aguado-Velasco and Bretscher, 1999Go).


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  		Table 1. Endocytosis by vegetative and starving cells

 

By contrast, starving cells take up much less fluid than growing cells. Growing cells take up FITC dextran linearly for around 40 minutes, before a plateau is approached when the rate of release equals that of uptake (Aubry et al., 1997Go). Aggregation-competent cells take up fluid at only 6% of this rate, confirming previous observations (Fig. 1C, Table 1).


Figure 2
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  		Fig. 2. Chemotaxis of temperature-sensitive NSF cells. Control Ax2 and temperature sensitive NSF cells (strain HM1067, nsfA2) were made aggregation-competent at 20°C, then shifted to the restrictive temperature of 28°C for at least 20 minutes, before being filmed moving towards a micropipette containing cAMP. The left-hand panels show stills from the films (Movies 1 and 2 in supplementary material) taken after 20-30 minutes. The insets show a typical elongated chemotaxing Ax2 cell and a rare nsfA2 cell that is chemotaxing efficiently but maintains a smooth profile and is only moderately elongated. The movement speeds of 68 (Ax2) or 80 (nsfA2) randomly chosen cells from each condition are tabulated in the right hand panels. Bar, 20 µm.

 
NSF is required for chemotaxis, but not for polarization of the leading edge
NSF is a soluble protein required for separating SNARE complexes and therefore essential for many membrane trafficking events (Malhotra et al., 1988Go). The nsfA2 temperature-sensitive allele of NSF is fast-acting and reversible. Vegetative cells carrying this mutation can barely move at the restrictive temperature of 28°C and endocytosis is blocked (Thompson and Bretscher, 2002Go). Aggregation-competent cells are similarly affected: membrane endocytosis is greatly reduced at 28°C (Table 1) and the cells rapidly round up and largely stop moving; the few that do move seem to shuffle, with greatly reduced pseudopodia (Fig. 2). When the mutant cells are restored to the permissive temperature, they resume movement within 20 minutes. In comparison, endocytosis and chemotaxis of wild-type Ax2 cells are unaffected at 28°C (see Movies 1-3 in supplementary material).

We tested whether nsfA2 cells could still be polarized by a cAMP gradient at the restrictive temperature. The first polarized responses are the rapid recruitment of PI 3-kinases to the cell cortex at the high point of the gradient, and the reciprocal loss of the phosphatase and tensin homolog (PTEN) phosphatase (Iijima and Devreotes, 2002Go; Funamoto et al., 2002Go), leading to an accumulation of PtdIns(3,4,5)P3 in the membrane. This in turn recruits PH-domain containing proteins to the plasma membrane (Parent et al., 1998Go; Meili et al., 1999Go). Almost simultaneously, actin is polymerized and a pseudopod projected.


Figure 3
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  		Fig. 3. Temperature-sensitive NSF cells can be polarized by cyclic-AMP gradients. Each panel shows temperature sensitive NSF cells (HM1067) expressing the appropriate GFP fusion proteins to act as reporters for cell polarization, were made aggregation-competent and stimulated with cAMP from a micropipette, at either the permissive temperature of 20°C (shown only for ABD-GFP) or the restrictive temperature of 28°C. Reporters: CRAC, reports PtdIns(3,4,5)P3 in the membrane; PTEN, normally bound to the membrane but repelled from the leading edge; ABD, binds to F-actin, thus reporting sites of actin polymerization. Wild-type cells behaved in a similar way at both temperatures, and did not round up at 28°C. When the pipette was moved to a new position (white circle) the image shown is the first taken at the new location; time in seconds after introducing the pipette is shown in each image; bar, 10 µm. See Movies 4-6 in supplementary material.

 
These events were reported in mutant cells using GFP-fusion proteins (Fig. 3; see Movies 4-6 in supplementary material). PI 3-kinase was recruited to the cortex adjacent to the micropippette within 5 seconds of it being introduced (not shown) and PTEN lost. A PH-domain reporter (Parent et al., 1998Go), which binds PtdIns(3,4,5)P3 in the plasma membrane, was also recruited in the mutant, essentially as in wild-type cells. Most significantly ABD-GFP, reporting F-actin (Pang et al., 1998Go), rapidly accumulates adjacent to the micropipette supplying cAMP, indicating extensive actin polymerization. At the permissive temperature, actin polymerization leads to expansion of a pseudopod, whereas at the restrictive temperature, it only causes a slight bulge in the membrane, or in extreme cases, no deformation at all: despite actin polymerization in a spatially correct manner, a pseudopod cannot be extended if membrane trafficking is inhibited.

Absence of detectable membrane flow from the leading edge
The membrane flow hypothesis proposes that exocytosis occurs predominantly at the leading edge of motile cells, and endocytosis randomly over the surface, thus creating a flow of membrane away from the leading edge, which could drive the cell forward (Bretscher, 1984Go). Such a flow might be detectable by tracking the movement of photo-bleached marks in the plasma membrane. These marks will be erased by diffusion of fluorescent molecules into them from the surrounding regions, so to be useful they must persist long enough for the cell to move an appreciable distance. If Dictyostelium cells move at 10 µm/minute and the mark is 2 µm diameter, then a lifetime of around 10 seconds should be sufficient. The diffusion coefficient of the cAMP receptor, cAR1, suggests this is feasible (Ueda et al., 2001Go).

We measured the first half-life of decay of spots bleached on the lower surface of non-motile cells expressing cAR1-GFP (Xiao et al., 1997Go), and found they ranged from 3 to 8 seconds. These spots did not move and decayed from the edge, consistent with recovery by diffusion of fluorescent molecules into the spot (see Movie 7 in supplementary material); the diffusion coefficient of cAR1-GFP calculated from the spot half-life is 4.7±1.5x10-10 cm2/second (n=8; each value is the mean of triplicate spots from the same cell), which is within a factor of two of the value obtained previously for cAR1 alone (Ueda et al., 2001Go). Vegetative cells gave a diffusion coefficient of 6.7±1.5x10-10 cm2/second (n=14).

Spots were bleached at different positions on the lower surface of moving cells, some by chance being adjacent to a position where a pseudopod subsequently formed (Fig. 4 and Movie 8 in supplementary material). It is apparent that these marks do not move backwards with respect to the leading edge as required by membrane flow, but in all cases they move forward, often keeping pace with it. Similar results were obtained with marks bleached on the top of cells (not shown). Marks in the rear of cells similarly moved forward, again keeping a roughly constant distant from the rear (Fig. 4, `Posterior'); those on immotile cells did not move at all.


Figure 4
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  		Fig. 4. Tracks of spots photobleached in the plasma membrane of moving cells. Spots were photobleached on the underside of cells expressing cAR1-GFP, and tracked until they faded. They were made either in the area where a pseudopod appeared (left and middle panels), or at the posterior of the cell (right panel `posterior'). The positions of the cells at various times is shown in the diagrams below. Graphs quantify displacement of the spot centre from the starting position in parallel with the leading edge (pseudopods) or the uropod (posterior) are shown at the bottom. Ax2 cells transformed with cAR1-GFP were made aggregation-competent and induced to migrate under a thin layer of agarose to constrain movement in the z-axis. Photobleaching was for 0.16, 0.79 or 1.32 seconds, giving spots of initial diameter 2.5 µm (pseudopods) or 2.0 µm (uropod); the frame rate was 1 frame per 0.8, 0.6 or 0.5 seconds; times are given in seconds; bar, 10 µm. See Movie 8 in supplementary material.

 
Cells were not visibly affected by photobleaching, but to increase confidence in the results, we made a photoactivatable version of cAR1-GFP (Patterson and Lippincott-Schwartz, 2002Go), because less energy is required for activation than bleaching. Photoactivated marks behave similarly to photobleached marks - they move forward with respect to the substratum, roughly in step with the leading edge (Fig. 5; Movie 9 in supplementary material). We also used a GFP derivative of a second membrane protein - SadA (Fey et al., 2002Go) - which was less satisfactory because of additional labelling of internal vesicles, but we still found that photobleached marks moved forward roughly with the leading edge (Movie 10 in supplementary material).


Figure 5
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  		Fig. 5. Track of a spot photoactivated in the plasma membrane of a moving cell. A spot was photoactivated on the underside of a cell expressing the cAMP receptor coupled to photoactivatable GFP and followed as a pseudopod extended. Ax2 cells transformed with plasmid pDT18 were made aggregation-competent and induced to migrate under a thin layer of agarose to constrain movement in the z-axis. Cell outlines were traced from the accompanying DIC image. Photoactivation was for 0.77 seconds and frames were taken every 0.59 seconds; times are given in seconds in the images; scale bar is 10 µm. To aid visualization, the contrast/brightness of these images was altered in ImageJ by conversion to 8-bit then changing the min/max pixels from 0-255 to 11-137. See Movie 9 in supplementary material.

 
Though we cannot detect any flow of membrane backwards from the leading edge of motile Dictyostelium cells, it remains possible that some membrane is inserted there, as might be the case for the cell shown in the middle panels of Fig. 4 where the bleached spot moves in the same direction as the leading edge, but lags behind it.

Surface area changes of cells moving under agarose
Dictyostelium cells continuously change their shape as they move, which most likely means that they also change their surface area. To determine whether this supposition is correct, we measured surface areas from three-dimensional (3D) reconstructions produced from confocal stacks of cells expressing cAR1-GFP. This protein is almost entirely restricted to the plasma membrane and reveals even fine filopodia projecting from the surface (Fig. 6A,B). The strongly fluorescent outline of an expressing cell is readily recognized using commercial software, allowing the surface to be reconstructed and its area measured (Fig. 6C,D; viewed from +30° and -30° to the horizontal). Since filopodia are often truncated in these reconstructions, their length was measured manually and their surface area calculated assuming they are cylinders of 0.1 µm diameter. To simplify the geometry, and reduce the number of confocal sections per stack, we initially studied cells moving under agarose, where they are reduced to a pancake shape of around 6 µm thick, with a smooth bottom and lumpy top. The statistical significance of membrane area changes was assessed by taking bursts of three confocal stacks (lasting 2-3 seconds) every 25 seconds and treating them as repeat measurements at a single time point. Since cells move slightly during these bursts, errors are overestimated and the significance of any differences correspondingly underestimated.


Figure 6
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  		Fig. 6. Surface reconstruction of a cell moving under agarose. Ax2 cells expressing cAR1-GFP were induced to chemotax under 0.6% agarose containing cAMP. Stacks of confocal sections were taken at the indicated times and reconstructed using the Volocity package. (A) A single confocal section through a cell at different times; (B) a maximum density projection of the z-section stack; (C) the 3D reconstruction viewed from 30° to the substratum; (D) the 3D reconstruction viewed from -30° to the substratum. The relative surface area and volume (A/V) of the cell are given in C. See Movie 11 in supplementary material.

 

In the typical cell shown chemotaxing towards cAMP in Fig. 7 and Movie 11 in supplementary material, the surface area of the cell body (filopodia truncated) increased by 15% over the first minute, as it extended a new pseudopod, and then gradually declined again as one pseudopod becomes dominant and the other retracts. The increases in surface area at 0-32 seconds and 32-69 seconds are each statistically significant, as is the decrease at 69-174 seconds (P<0.001, one way ANOVA with Tukey's test). The cell carries between 5 and 14 filopodia in this period, but their combined area never exceeds 1.5% of the cell body, and is actually at its peak when the cell body area also peaks at 69 seconds. Cell volume fluctuated by no more than 1.6% over the whole period, with no change being statistically significant (P>0.05).


Figure 7
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  		Fig. 7. Surface area and volume of a cell moving under agarose. Ax2 cells expressing cAR1-GFP were induced to chemotax under 0.6% agarose towards cAMP. Bursts of three sets of confocal stacks (12 sections taking three seconds) were taken at approximately 25 seconds intervals and the areas and volumes obtained from each burst treated as repeat measurements at the same time point, thus allowing statistical analysis (1-way ANOVA, with Tukey's test). (A) Relative surface area and volume of the cell body (without filopodia); (B) number and total length of filopodia, measured manually; (C) relative surface area of cell body and filopodia (assumed to be 0.1 µm in diameter); plotted to the same scale, but note the different origins. The increase in surface area at 0-69 seconds and the subsequent decline at 69-174 seconds are both significant at P<0.001, whereas none of the volume changes are (P>0.05). The cell is the same one as shown in Fig. 6 with an initial surface area of the body of 698 µm2 and of the filopodia of 3.5 µm2 (0.5 % of the total surface area); and a volume of 586 µm3.

 

A total of 10 cells analysed for 4-10 minutes all produced statistically significant changes in cell body surface area (P<0.001), with the largest increases for each cell ranging from 4.9-20.7% (mean 9.6±4.8%; ±s.d., n=10) and the largest decreases from 3.3-17.1% (mean 10.9±4.8%; ±s.d., n=10). Filopodia contributed 0.29-2.1% of surface area (mean 0.91±0.37%; ±s.d., n=10) and thus their withdrawal and extension cannot provide sufficient surface area to account for the observed fluctuations in cell body surface area.

In some cells there were also statistically significant changes in volume with increases of up to 8.3% and decreases of up to 12.3% (P<0.001). However, surface area expansions were often observed without a significant volume expansion, indicating that the two processes are not tightly coupled.

Surface area changes of unconstrained cells
We performed similar experiments with cells moving freely under buffer and expressing soluble GFP, which gives good surface rendering, but was less good for visualizing filopodia than cAR1-GFP. Since these cells are more rounded than those under agarose, reaching a height of 10-15 µm above the substratum, more sections were required in each confocal stack, and it was impractical to treat successive stacks as duplicates.

Fig. 8 shows the morphology of individual cells viewed from 30° to the horizontal, together with their relative areas and volumes (Movies 12-14 in supplementary material). Randomly moving cells change dramatically in shape and in relative area, especially when they round up and flatten out. The one shown in Fig. 8A is relatively elongated at the start of the sequence, and then goes through two cycles of rounding (at 60 and 260 seconds) and flattening during the next 12 minutes. As the cell flattens at 260-640 seconds, the surface area increases by 31%, whereas in the succeeding 80 seconds it rounds up and shrinks towards its original area. The chemotacting cell shown in Fig. 8B maintains a more constant shape, though with surface area fluctuations of 12% over 200 seconds. We also used a protocol where cells were first induced to round up by chilling on ice for 10 minutes, and then induced to move by warming to 22°C and stimulation with a micropipette containing cAMP. The cell in Fig. 8C rapidly flattens out and advances to the micropipette, embracing it at 140 seconds with an area increase of 20%. By contrast, there is little change in the surface area of the quiescent cell shown in Fig. 8D.


Figure 8
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  		Fig. 8. Surface area and volume of cells moving under buffer. Ax2 cells expressing soluble GFP were brought to aggregation-competence and filmed by taking confocal stacks every 9-13 seconds; the reconstructed cells are viewed at a 30° angle to the substratum. The surface area and volume of the cell are given above each image (A, V, respectively), normalized to the values at the start of the sequence.(A) A randomly moving cell (at 0 seconds the surface area=567 µm2; volume=547 µm3); (B) chemotaxing cell moving to the right towards a micropipette containing cAMP (506 µm2; 562 µm3); (C) a quiescent cell, stimulated with a micropipette containing cAMP (to the right) at the start of the sequence (397 µm2; 361 µm3); (D) a quiescent cell, not stimulated (211 µm2; 191 µm3). Bars, 15 µm. Selected time points are shown. See Movies 12-14 in supplementary material.

 

Surface area measurements from a number of cells are summarized in Fig. 9. Both randomly moving and chemotaxing cells show substantial surface area fluctuations, which were greater in randomly moving cells, with increases of up to 35%, than in chemotaxing cells (25%) due to their greater propensity to round up and flatten out. Rounded cells that are stimulated with a cAMP-filled micropipette consistently show an increase in surface area of around 20%, whereas rounded, quiescent cells change very little.


Figure 9
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  		Fig. 9. Quantification of cell surface area changes during movement under buffer. Aggregation-competent cells, expressing soluble GFP, were filmed by taking confocal stacks at 15-30 second intervals. Changes in relative surface area of representative cells (A) moving randomly; (B) chemotaxing steadily towards a micropipette containing 1-2 µM cAMP; (C) quiescent, but stimulated to move at the start of the sequence with a micropipette containing cAMP; (D) quiescent, but unstimulated.

 

   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Conclusion
 Materials and Methods
 References
 
In this paper, after confirming that moving Dictyostelium cells maintain an active endocytic cycle and that movement depends on an active NSF membrane trafficking protein, we investigated three possible roles that the endocytic cycle could play in cell motility: to help polarize the plasma membrane during pseudopod formation, to create membrane flow and to allow the cell to alter its surface area as it changes shape.

Endocytosis by aggregation-competent cells
Aggregation-competent cells in suspension take up an area equivalent to their entire surface every 11 minutes, comparable to the rate of growing cells (Aguado-Velasco and Bretscher, 1999Go). The first phase of FM1-43 uptake rapidly saturates implying that most membrane returns to the surface within about 5 minutes, consistent with previous surface labelling experiments (Neuhaus and Soldati, 2000Go). If the primary endocytic vesicles are spherical, their mean diameter can be estimated, from the rates of volume and surface uptake, as around 50 nm (see Materials and Methods). This is somewhat smaller than Dictyostelium coated vesicles (Swanson et al., 1981Go) and early endosomes (Neuhaus et al., 2002Go) but in the range of other small trafficking vesicles, such as synaptic vesicles (Takamori et al., 2006Go). It seems that aggregation-competent cells rapidly endocytose their plasma membrane in small vesicles, and return it to the surface within a few minutes.

By contrast, cells growing in axenic medium take up most of their fluid by macropinocytosis in much larger vesicles of more than 1 µm in diameter (Hacker et al., 1997Go), and only return it to the medium after a 45 minute lag (Aubry et al., 1997Go).

Movement, but not initial cell polarization, depends on a functional NSF protein
NSF is required for disengaging SNARE complexes and is thus essential for many steps in the endocytic cycle (Malhotra et al., 1988Go). The nsfA2 temperature-sensitive allele of NSF is fast-acting and fully reversible. We find that endocytosis and movement of starving, mutant cells are largely blocked at the restrictive temperature, as previously reported for growing cells (Thompson and Bretscher, 2002Go). Polarization of the PtdIns(3,4,5)P3 signalling system (PI 3-kinases, PTEN and PtdIns(3,4,5)P3 itself) is unaffected in these conditions, suggesting that it does not depend on membrane trafficking, such as localized exocytosis to the leading edge. These cells round up at the restrictive temperature, and their surface area shrinks by around 25% (D.T. and R.R.K., unpublished observations), so it is likely that the physical expansion of pseudopodia is constrained by an insufficiency of membrane, even though actin polymerizes at the expected `up-gradient' site of pseudopod formation.

Absence of detectable membrane flow from the leading edge
Membrane flow from the leading edge of moving cells was suggested to explain the rearward movement of particles bound to the surface of many cell types (Abercrombie et al., 1970Go). It was proposed to be produced by localized exocytosis at the leading edge and to provide motive force to the cell (Bretscher, 1984Go). Results consistent with membrane flow have been obtained from giant HeLa cells (Bretscher, 1983Go) fibroblasts (Hopkins et al., 1994Go; Schmoranzer et al., 2003Go) and Physarum plasmodia, where membrane vesicles can be seen approaching and fusing with the leading edge (Sesaki and Ogihara, 1997Go). However, experiments with leukocytes and keratocytes, failed to detect any significant rearward membrane flow (Lee et al., 1990Go; Kucik et al., 1990Go), suggesting that this is not a universal property of moving cells, though it may be important for some of them.

We tracked photobleached or photactivated registration marks in the plasma membrane, with respect to the leading edge of a pseudopod. In no case did they move backwards from the leading edge, as required by the membrane flow hypothesis, but usually they moved forward roughly in concert with it. It might be argued that photobleaching somehow alters pseudopod behaviour; however, we saw no obvious morphological response to photobleaching and obtained a similar result from photoactivation experiments, where the light dose was reduced. We conclude that Dictyostelium cell movement does not involve any detectable membrane flow from the leading edge in the conditions we have used, consistent with the finding that late endosomes fuse with the plasma membrane preferentially at sites removed from pseudopodia (Charette and Cosson, 2006Go). It seems that membrane required for the expansion of a pseudopod is not exocytosed at the leading edge but may be added at less defined sites in the cell body, and then `dragged' forward as required.

Surface area changes in cell movement
A variety of non-secretory cell types can rapidly and substantially increase their surface area by exocytosis of membrane. These include tissue culture cells stimulated by calcium transients (Borgonovo et al., 2002Go), phagocytocytic cells engulfing a particle (Holevinsky and Nelson, 1998Go; Hackam et al., 1998Go) and epithelial cells lining the bladder or lung alveolae, which must cyclically increase their surface area (Truschel et al., 2002Go; Fisher et al., 2004Go). Since moving Dictyostelium cells constantly change their shape, it is no surprise that they also change in surface area, often by 20-30% over a few minutes.

As membranes are barely stretchable - only a 2-3% area increase is possible before they rupture (Mohandas and Evans, 1994Go) - an increase in apparent surface area requires either exocytosis of internal membrane or the utilization of a reserve of folded surface membrane. Concertina-like membrane folding is seen in large amoebae (Komnick et al., 1973Go), but not in Dictyostelium (Maeda and Gerisch, 1977Go; Maeda and Eguchi, 1977Go; Neuhaus et al., 1998Go). A related possibility is that filopodia might provide a reserve of surface area (Erickson and Trinkaus, 1976Go), but we have eliminated this possibility by separate measurement of their area. Although we cannot completely exclude the possibility of a membrane reservoir undetectable by our methods, we prefer to propose that the measured changes in surface area result from changes in the balance of membrane exocytosis and endocytosis.

Electron microscopy reveals abundant vesicles in the cytoplasm of aggregation-competent amoebae, including a population of small vesicles whose number fluctuates when cells in suspension are stimulated with cAMP (Maeda and Gerisch, 1977Go). As their number drops, extensive fusion of vesicles to the plasma membrane is evident. These small vesicles are therefore a good candidate for the reservoir of membrane needed by moving cells to adjust their surface area.


   Conclusion
Top
 Summary
 Introduction
 Results
 Discussion
 Conclusion
Materials and Methods
 References
 
This work was stimulated by proposals in the literature linking cell motility and membrane trafficking and by the unexplained dependence of Dictyostelium cell movement on a fully functional membrane trafficking system (Wessels et al., 2000Go; Thompson and Bretscher, 2002Go). Moving cells maintain an active endocytic cycle, and experience substantial surface area changes as they change shape. We propose that one function of membrane trafficking is to allow these cells to change surface area, and thus shape; when there is an insufficiency of surface, as in the temperature-sensitive NSF mutant, movement is severely impeded.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Conclusion
 Materials and Methods
References
 
Cell biology and molecular biology
Cells were grown and studied at 22°C unless otherwise stated. Ax2 and the temperature sensitive mutant HM1067 nsfA2 (Thompson and Bretscher, 2002Go) cells were grown in suspension or tissue culture plates in axenic medium (Watts and Ashworth, 1970Go) with vitamins (0.1 mg/l B12, 0.02 mg/l biotin and 0.2 mg/l riboflavin; 20°C for HM1067). Aggregation competent amoebae were obtained by resuspending washed cells (2x107/ml) in KK2 (16.5 mM KH2PO4, 3.8 mM K2HPO4, 2 mM MgSO4), shaking at 180 rpm (20°C for HM1067) for 1 hour and pulsing with 70-90 nM cAMP every 6 minutes for 3-5 hours. Cells for cAMP stimulation were `basalated' with 5-7.5 mM caffeine for 20 minutes, to inhibit endogenous signalling, at either 20°C or 28°C, harvested, washed twice in KK2, resuspended at 2x107/ml in KK2 and the cells at the restrictive temperature of 28°C were used immediately; other cells were stored on ice until required.

Cells were transformed with the reporter constructs CRAC-GFP (Parent et al., 1998Go), GFP being driven by the actin 15 promoter (Gerisch et al., 1995Go), cAR1-GFP (Xiao et al., 1997Go) and a photoactivatable version of it (Patterson and Lippincott-Schwartz, 2002Go), ABD-GFP (Pang et al., 1998Go), actin-GFP (Westphal et al., 1997Go), PI 3-kinase-2 fused to cyan fluorescent protein (PI3K2-CFP) or PTEN-CFP (Funamoto et al., 2002Go), by electroporation with 20-30 µg of plasmid DNA per 4x106 cells (Knecht and Pang, 1995Go). Transformants were selected and maintained in axenic medium supplemented with 20 µg/ml G418, or 10 µg/ml blasticidin for plasmid pDT18. Mixed populations were used, except in the case of Ax2 expressing GFP, where a stable, strongly expressing clone was selected (strain HM2068).

Plasmid pDT18 (photoactivatible GFP fused to the C terminus of cAR1): cAR1 coding sequence, including the 5' ribosome-binding site, was PCR amplified from pZX2 cAR1-GFP (Xiao et al., 1997Go) (5' primer: 5'-CAAAAGCTTAAATCGAATAAATTTAAGATTTTTCACAC-3'; 3' primer: 5'-CTTCTCCTTTACTCATGAATTCATTATTTCCTTGACC-3'), the product ligated into pBluescriptII KS (HindIII-EcoRI digestion) and photoactivatible GFP (Patterson and Lippincott-Schwartz, 2002Go) PCR amplified from pcDNA3 (from Ben Nichols) (5' primer: 5'-CCGGAATTCAAAATGGTGAGCAAGGGCGAGGAGCTGTTCACC-3'; 3' primer: 5'-GTTGGATCCTTACTTGTACAGCTCGTCCATGCCGAGAGTG-3') ligated to cAR1 in pBluescrpitII KS (EcoRI-BamHI digestion). CAR1-photoactivatible GFP fusion was excised with HindIII-BamHI and inserted into the same sites of the Dictyostelium vector mRFPmars (Fischer et al., 2004Go), yielding pDT18.

Microscopy and image analysis
Images of cells in Lab-Tek (Nalgene) chambered coverglasses containing 2 ml of KK2 were obtained using a 60x 1.4 NA oil immersion lens on a Nikon Eclipse TE300 inverted microscope, with a Bio-Rad Radiance confocal system. Temperature was controlled using a heated microscope stage (Linkam MC60) and an ASI 400 Air Stream Stage Incubator (Nevtek); cells were stimulated with a micropipette (Eppendorf Femtotips II) containing 1-2 µM cAMP using a micromanipulator (Eppendorf 5171). Immotile, rounded cells were produced by placing the coverslip on ice for 10 minutes. For under-agar experiments (Laevsky and Knecht, 2001Go) 0.3-0.5 ml 0.6-0.7% SeaKem GTG agarose in KK2 was added per coverglass chamber and a 2x15 mm trough was cut and filled with 25 µl of KK2 containing 1-5x104 cells. Cells crawled under the agar by random movement, or chemotaxis after doping the agar with 1 µM cAMP. After 1-2 hours, when sufficient cells had migrated under the agar, 400 µl of KK2 was added to each chamber to prevent desiccation.

Cell velocity was measured using the manual tracking plug-in of ImageJ (Wayne Rasband, NIH). Surface area and volume were measured from reconstructions of stacks (0.3 or 0.5 µm increments) from strains expressing cAR1-GFP or GFP alone (HM2068) using Volocity (Improvision). z-axis elongation, due partially to mismatch in refractive indices, was determined by comparing the z- and x/y-axes of fluorescent beads (9.7 µm diameter FluoSpheres; Molecular Probes) and corrected by dividing stack increments by 1.97 (giving true z-axis increments 0.15 and 0.25 µm). Successive estimations of surface area and volume of a given bead differed by <1% and volumes were within 10% of that calculated from diameters. Surface area is systematically over-estimated by Volocity: the manufacturer reports that the surface area of a perfect sphere is over-estimated by 25% and we found the surface area of beads was 35% greater than calculated from their diameter. However, our conclusions depend on comparative, not absolute, surface areas and are qualitatively robust under varying conditions of microscopy (thickness of optical sections), and image analysis (smoothing of the image and selection of perimeter-recognition parameters). Filopodia visible in the raw images, tend to be truncated by the perimeter-recognition software; their individual length was measured separately and area calculated assuming a diameter of 0.1 µm.

GraphPad Prism 4 was use for statistical calculations.

Photobleaching and photoactivation
We employed a Zeiss LSM510 META confocal microscope with 40 mW argon (514 nm and 488 nm lines), 5 mW HeNe (546 nm line), and 80 mW krypton (413 nm line) lasers, collecting 16-bit images with a 63x 1.4 NA objective. cAR1-GFP was bleached at 488 nm (0.3-1.5 seconds, maximum power) and imaged using the 488 nm line (every 0.5-1.3 seconds, 5-15% maximum power). Photoactivatable GFP was activated at 413 nm (0.2-1.0 seconds, 40% maximum power) and imaged at 488 nm (every 0.59 seconds, 5-15% maximum power). Marks were 1.5-3.5 µm diameter.

Images were analyzed with the LSM reader plug-in of ImageJ. Intensity in regions representing the background (A), the entire cell (B) and the photobleached region (C) was measured and the ratio (C-A)/(B-A) normalized to the first pre-bleached frame, after setting the first bleached frame to 0. Recovery was fitted to a single exponential curve to obtain the half-time of fluorescence recovery (t1/2). Diffusion coefficients were calculated using D=(0.224xr2)/t1/2 where r is the radius of the photobleached region in cm (Soumpasis, 1983Go).

Endocytosis
Membrane uptake: 5 ml cells at 2.5x106/ml in MK (20 mM MES, 10 mM KCl pH 6.2) were shaken at 180 rpm. At t0, 10 µM FM1-43 (a lipophilic dye taken up by membranes) was added and the fluorescence measured (PerkinElmer LS50 fluorimeter: 470 nm excitation, slit width 5 nm; 570 nm emission, slit width 10 nm) within 8 seconds, and the cells were then returned to the flask. In later experiments, uptake was measured directly with cells in a stirred cuvette; but note that this gives a higher uptake rate for reasons unknown.

Fluid phase uptake: FITC-dextran (Sigma FD-70; 70 kDa; 2 mg/ml final) was added to cells (1x107/ml) in MK which were shaken at 180 rpm, and triplicate 0.8 ml samples were taken into 0.75 ml ice-cold MKB buffer (MK buffer plus 0.5% w/v BSA) in a microcentrifuge tube held on ice. Cells were pelleted, the supernatant aspirated, and the pellet washed twice more in 1.5 ml ice-cold MKB, lysed in 1 ml 100 mM Tris-HCl, 0.2% (v/v) Triton X-100, pH 8.5 with agitation on a vibramixer for 5 minutes, briefly centrifuged (16,100 g, 2 minutes) and the supernatant fluorescence was determined (excitation 490 nm, slit width 2.5 nm; emission 520 nm, slit width 10 nm). Control samples were chilled in iced water for 10 minutes before adding FITC-dextran.

Size of primary endocytic vesicles
Endocytic vesicle mean diameter was calculated from the rates of volume uptake and surface uptake using the relationship V/A=r/3. As area uptake is measured relative to total area, an estimate of the total surface area of the cell is required. Reconstructions of aggregation-competent cells on a surface, give an uncorrected value of 694±180 µm2 (n=31), which is reduced to 452 µm2 because of the over-estimation of surface area given by the Volocity program (see above). Using this corrected value, the internal diameter of the endocytic vesicle (assumed to be round) is 41 nm. Allowing 4 nm for each membrane, the outer diameter of the vesicle is 49 nm. If cells are more rounded in suspension, where the uptake measurements are made, than on a surface, where the surface area is measured, the surface area may be an overestimate and the diameter correspondingly underestimated. The volume of these cells is 646±258 µm3 (n=31), which could be contained in a sphere of diameter 10.7 µm and area 361 µm2, giving an external diameter of 60 nm for the primary endocytic vesicle. Note that estimates are sensitive to any deviation from the assumption that FM1-43 uptake follows `average' membrane uptake - for instance if it was somewhat enriched in, or excluded from, the primary uptake vesicles.


   Acknowledgments
 
We are grateful to Mark Bretscher for interesting us in his ideas on the involvement of the plasma membrane in movement and for the nsfA2 strain, the Dictyostelium group for discussions and Ben Nichols for help with the photobleaching experiments.


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/14/2318/DC1


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 Introduction
 Results
 Discussion
 Conclusion
 Materials and Methods
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