MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
* Present address: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Author for correspondence (e-mail: msb{at}mrc-lmb.cam.ac.uk)
Accepted 13 June 2002
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SUMMARY |
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Key words: NSF, Cell polarity, Cell locomotion, Endocytosis, Drosophila, Dictyostelium
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INTRODUCTION |
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The leading edge of a cell is believed to contain the motor that drives locomotion and pulls the rest of the cell forwards, although how this motor works is debatable. In migrating fibroblasts, both exocytosis (Bretscher, 1983; Bretscher and Thomson, 1983
) and actin polymerisation occur here (Wang, 1985
), whereas crosslinked antigens form a cap at the trailing end of the cell (Taylor et al., 1971
). This polarity is presumed to be a reflection of the existing arrangement of the actin and/or microtubule cytoskeletons. In a migrating cell, this internal polarity can be extremely flexible, as demonstrated by the ease and speed with which a cell can reorientate in the face of a changing chemotactic signal (Parent and Devreotes, 1999
). Understanding how external signals cause a cell to reorientate may provide a route to unravel the internal machinery which defines the polarity of a cell and how the motor works. Much progress is being made in the highly motile amoeba of Dictyostelium discoideum to define those genes whose products guide the processes which lie between chemoattractant and chemotaxis (Chung et al., 2001
). However, such genetic studies are limited at present to the disruption of non-essential genes by homologous recombination (De Lozanne and Spudich, 1987
; Manstein et al., 1989
). Many genes whose products might have been expected to be required to function in the motor of the cell can be inactivated, usually giving a surprisingly normal cell (Noegel and Schleicher, 2000
). It therefore seemed desirable to develop methods for making conditional mutants in essential Dictyostelium genes, as is routinely done in yeast.
Dictyostelium amoebae have a haploid genome; this makes it possible to replace an individual gene with a disrupted copy by homologous recombination. We hoped to adapt this procedure to replace an essential gene with a copy containing a mutation that would make the encoded protein temperature sensitive (ts). For this, we chose the gene for NSF for a variety of reasons. First, NSF is required to dissociate snare complexes after membrane fusion has occurred (Beckers et al., 1989; Malhotra et al., 1988
; Whiteheart et al., 2001
). As such, it would be expected to provide an essential function and, unsurprisingly, null mutants in yeast (Sec18) and Drosophila (comatose) are lethal. In Dictyostelium, there is a single gene for NSF (nsfA), the cDNA sequence of which has been determined (Weidenhaupt et al., 1998
). Second, the amino acid sequence of NSF is highly conserved, allowing point mutants in one animal to be transplanted into another. In the comatose gene several ts mutants exist whose amino acid exchanges are known (Pallanck et al., 1995
; Siddiqi and Benzer, 1976
; Tolar and Pallanck, 1998
). Third, the behaviour of a ts mutant in nsfA might shed light on the role of membrane circulation in cell locomotion (Abercrombie et al., 1970
; Bretscher, 1984
).
This straightforward approach of exchanging an amino acid in nsfA to make a ts mutant did not succeed. However, the high level of homologous recombination we found at this locus made it feasible to replace the endogenous nsfA with a PCR mutagenised nsfA library and so generate a panel of ts mutants. We analysed two NSF ts mutants and find that they are unable to carry out normal endocytic processes at the restrictive temperature. In addition, they appear to have lost their cell polarities and are consequently unable to move.
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MATERIALS AND METHODS |
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pC4
The DNA sequence around the mutated site was changed from GCC.AGA.CAA.ATC.GGT.AAG.ATG.TTA.AAT to .GCG.CGC. CAA.ATC.GAA.AAG.ATG.TTA.AAT, introducing a unique GCGCGC (BssHII) site.
pC6
Likewise, the sequence was altered from GAG.ATC.TCA. TTA.CCC.GAT.GAA.CAT to GAG.ATC.TCC.TTA.AGT.GAT. GAA.CAT, introducing a unique CTTAAG (AflII site).
pC6N
A nonsense codon was placed in frame with the Com6 mutation so that the relevant sequence was GAG.ATC.TCC.TTA.AGT. TAA.GAA.CAT
Positions of all sites are given from the 5' end of the construct, defined by a PstI site. Two libraries were used in which the DNA had been mutagenised between bases 1140-1700 (coding largely for the D1 domain of the protein; library size 60,000) and bases 1700-2860 (coding for the last 60 residues of D1 and the whole of D2; library size
6000). These were incorporated into pC6 and yielded the mutant lines ts4, ts5 and ts7, and ts1-3, ts6 and ts8-11, respectively.
The mutagenised regions from the two mutants described here were copied out of genomic DNA and two independent clones of each were sequenced. The mutations are: in nsfA1, A1847G (giving amino acid change K472E), A1939T (E502D), T1977C (V515A), T2005G (N524K), A2025T (K531M) and A2366T (I645F); and in nsfA2, A1854T (E474V), T1984A (D517E), C2131A (F566L), A2398T (K655M) and T2574A (M714K). In addition, these lines also carry the Com6 mutation.
Cell transformation and mutant selection
DNA was linearised and introduced into Ax2 amoebae by electroporation (Howard et al., 1988). Cells were then diluted into medium at 3x105/ml and dispensed into flat-bottomed 96-well plates at 0.1 ml/well and held at 20°C. After 1 day, 0.05 ml 30 µg/ml blasticidin in medium was added/well and the cells grown until colonies were apparent. During this selection process, all transfected cells start with a complement of wild-type NSF and, as such, would initially be expected to grow normally. If the endogenous gene were replaced by one encoding a poorly functional NSF, the clone might grow more slowly at the permissive temperature once the native NSF had become depleted; indeed, it might grow only by phagocytosis (of its dead neighbours) and not axenically, and thus give rise only to small clones. Although likely to be clonal, the content of each well was recloned: the clone size was guessed (between 50-3000 cells) and an aliquot of
30 cells plated out on bacteria at 20°C. Two colonies from each plate were tested for growth on bacteria at 20°C and at 26.5°C. Temperature-sensitive clones were saved. The strain harbouring the Com6 mutation is HM1058; the original nsfA 3, 4-9 and 10,11 lines are HM1059, 1061-1066, 1068, 1069. The recreated nsfA 1 and 2 lines are HM1060 and HM1067, respectively. HM1060 can only be grown on bacterial plates, whereas HM1067 will grow on plastic or in suspension in axenic medium.
Endocytosis assays
FITC-dextran
Cells (2.5x107/ml) were preincubated in KK2, FITC-dextran (to 4 mg/ml) added and aliquots removed at the times indicated, washed with cold buffer containing 1% FCS, dissolved in detergent and the fluorescence measured (Aguado-Velasco and Bretscher, 1999).
FM1-43
Cells (2.5x107/ml) were preincubated in KK2, FM1-43 added to 10µM and dye uptake measured (Aguado-Velasco and Bretscher, 1999) but in the absence of added sorbitol, as some cell lysis occurs with sorbitol with both ts1 and ts2 at 28°C; this results in a high background and poor recovery of cells. The rate of uptake of FM1-43 by bacterially grown cells is unaffected by sorbitol. Unlike measurements on FITC-dextran uptake, FM1-43 uptake is extremely sensitive to cell damage as the dye binds tenaciously to intracellular components, presumably nucleic acids. It is possible that some of the apparent uptake of dye at 28°C is caused by cell damage, although inspection of labelled cells suggests this is not the case.
Phagocytosis
This was measured using fluorescent 1 µm beads (Witke et al., 1992).
Microscopy
To examine the effect of high temperature on the behaviour of mutant cells, a slide was constructed whose temperature could be rapidly changed. It had two chambers: an upper one on that an inverted coverslip with attached cells is held over a drop of buffered salt solution (KK2) by 1 mm high glass posts; and a lower sealed chamber separated from the upper chamber by a coverslip. This lower chamber, with a thickness of 2 mm, has attached entrance and exit tubes through which water can flow. This allows the temperature of the upper chamber, monitored with a small thermistor, to be changed from 20°C to 28°C within about 15 seconds. The slide was set up on a BioRad Radiance confocal microscope with a 10x air objective and DIC images collected.
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RESULTS |
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We decided to focus on just two mutants, ts1 (nsfA1) and ts2 (nsfA2), because, on warming to a restrictive temperature of 28°C, both showed early and similar phenotypic changes, yet only one (ts2) was able to grow in liquid culture (axenic growth). That they are indeed true gene replacements is shown by Southern blots (Fig. 1C): both lines also carry the Com6 site (Fig. 1D). To prove that the temperature sensitivity of each of these two lines is caused by mutation in nsfA, we cloned each mutated region (NsiI to BamHI, Fig. 1A) by PCR. Their sequences showed that ts1 and ts2 contained, respectively, six and five induced mutations that affect the coding region. These mutated regions were reassembled into the standard pC6 vector (replacing the wild-type NsiI/BamHI sequence) to generate two new mutagenic vectors. These were transformed into Ax2: both vectors gave rise to a high proportion of ts clones. This shows that it is possible to recreate these ts mutants with mutated DNA, and therefore that the mutations that cause temperature sensitivity are indeed in nsfA.
As mutations in NSF would be expected to affect membrane processes, we first examined endocytosis. Recreated lines were used and, unless otherwise stated, were harvested from bacterial plates.
Endocytosis
Three assays have been used to measure different aspects of surface uptake by amoebae: medium (fluid phase) uptake, cell-surface uptake using the dye FM1-43 and phagocytosis.
For fluid phase uptake, intracellular accumulation of FITC-dextran from the medium was measured (Hacker et al., 1997; Kayman and Clarke, 1983
; Vogel et al., 1980
). Cells were preincubated at either 22°C or 28°C for 20 minutes, the fluorescent tracer added and uptake followed with time (Fig. 2A). At 22°C, Ax2 and the ts lines have similar rates of fluid uptake; by contrast, at 28°C, this uptake is severely depressed in the ts lines and enhanced in Ax2. This shows that macropinocytosis in the mutants is temperature sensitive. However, the apparent similarity in the rate of FITC-dextran uptake in each line at 22°C was somewhat surprising, as ts2, but not ts1, is able to grow axenically. As axenic growth results in, and requires, a five- to tenfold increase in the rate of fluid phase uptake (Aguado-Velasco and Bretscher, 1999
; Kayman and Clarke, 1983
), we also determined the rate of FITC-dextran uptake with cells grown in axenic medium (Fig. 2B). Cells were harvested from bacterial plates, washed free of bacteria and incubated overnight in axenic medium. Under these conditions, both Ax2 and ts2 show a similarly enhanced rate of fluid phase uptake at 22°C, whereas ts1 does not: ts1 appears unable to be upregulated, the low levels of uptake being the same as when the cells are grown on bacteria. As macropinocytosis is used to take up nutrients for axenic growth, this observation provides a simple explanation for the lack of growth of ts1 in axenic medium, although how an apparently inductive process can be affected by mutation in NSF is intriguing.
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Phagocytosis was measured by the uptake of fluorescent microbeads (Fig. 2D) (Vogel et al., 1980; Witke et al., 1992
). At 22°C, both ts lines accumulate beads at about half the rate seen with Ax2; at 28°C, the rate of accumulation of beads by Ax2 remains about the same, but that of the ts lines drops at least 20 times to levels indistinguishable from background. As the ts lines grow more slowly than Ax2 on a lawn of bacteria at 22°C, this is consistent with the decreased rates of phagocytosis observed at the permissive temperature compared with Ax2. Furthermore, as both ts lines do not phagocytose at the restrictive temperature, this may explain why they do not grow on a bacterial lawn at 28°C.
The endocytic phenotypes of the two mutants, when bacterially grown, are very similar. All the membrane processes we examined fluid phase uptake, internalisation of cell surface area and phagocytosis are temperature sensitive. Although this is anticipated if NSF is required in at least one membrane fusion step in these different membrane cycles (Beckers et al., 1989), the extent of sensitivity does not seem to be the same for each cycle and this was not anticipated. In particular, fluid phase internalisation and phagocytosis are inhibited by about 95-100% after 20 minutes uptake at 28°C, yet surface uptake, measured with FM1-43, is only inhibited by about 75% (both normalised with respect to Ax2) and even less when shorter times are compared (Fig. 2C). We believe that this difference is significant and may mean that the cycle in which surface uptake occurs the molecular basis of which is unknown may not be directly dependent on NSF. By contrast, the endocytic cycles responsible for fluid phase uptake and for phagocytosis presumably do depend on NSF and at 28°C are blocked quickly. The uptake cycle by which FM1-43 accumulates inside the cell may depend on an NSF homologue, such as p97 (Muller et al., 1999a
), and slows down at 28°C as NSF-dependent trafficking processes cease.
Cell locomotion and capping
With the assurance that these mutants are defective in endocytosis, we next sought to discover how cell locomotion may be affected and therefore what the role of endocytosis may be in motile processes. Initially, we examined the ability of Ax2 and the mutants to chemotax towards cAMP; this was examined in a drop assay on an agar surface performed over 3 hours. Although all three lines chemotaxed at 22°C, only Ax2 did so at 28°C; cells in both mutants became round and, over the period of the experiment, no noticeable taxis occurred (C. R. L. T. and M. S. B., unpublished). Ax2 cells continue to move normally and ts1 cells behave like ts2.
As this deficiency could lie in the inability of the cells either to recognise chemotactic signals or to move, we examined cell locomotion in a slide chamber whose temperature could be controlled. This revealed that cells of both lines appear to move normally at 22°C; however, about 10 minutes after a temperature shift to 28°C, cells of both lines (unlike Ax2) start to become rounder and largely cease moving after a further 10 minutes (Fig. 3; see Movie 1 at http://www.mrc-lmb.cam.ac.uk/PAL/Sup_Material_MSB). Rounding up of ts cells might be caused by different steps in membrane circulation stopping at different times after a temperature shift. For example, if endocytosis continued after exocytosis had ceased, the net loss of surface area might round the cells up. To examine this, ts1 cells were held at 28°C, and thin sections were prepared and examined by electron microscopy. This showed that the surfaces of these cells are far from taut: they usually have a scalloped appearance, which suggests that the rounding up was not caused by a lack of surface area (not shown). Although these cells do not translocate, they are not frozen in shape: some residual motion remains due to the frequent protrusion of bulges, giving the cells a rabbit in a sack appearance, as seen in Movie 2 (http://www.mrc-lmb.cam.ac.uk/PAL/Sup_Material_MSB). These bulges may reflect nuclear motion or exocytosis of that membrane associated with the remaining surface uptake that still seems to occur at the restrictive temperature. Our observations indicate that membrane circulation and cell locomotion are connected processes, although they do not show what that connection is.
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Cell polarity and the cytoskeleton
The mutant lines described here represent the first molecularly defined mutations which block cell locomotion. They show that cap formation, like cell migration, is dependent on the continued activity of NSF. But how these motile processes are linked to NSF is unclear. As locomotory processes clearly depend on the actin cytoskeleton, we examined the distribution of F-actin in mutant cells under normal and restrictive conditions. ts2 cells were held at either 20°C or 28°C before fixing, permeabilising and staining with fluorescent phalloidin to locate F-actin (Fig. 5B). This shows that, at 20°C, F-actin is localised at the leading edge and in foci. However, in the spherical cells seen at 28°C, although the amount of F-actin is unaffected by the temperature change (normalised to Ax2; see legend to Fig. 5), it is rearranged so that the inner surface appears fairly uniformly covered, although randomly placed foci still exist (Fig. 5C; see Movie 3 at http://www.mrc-lmb.cam.ac.uk/PAL/Sup_Material_MSB).
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DISCUSSION |
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For a cell to translocate across a substrate, two conditions need to be satisfied: the cell must have a functional motor and the motor must have a polarity. Our results show that, at high temperature, a mutant cell stops translocating and this implies that either the motor or the polarity of the cell (or both) is defective. The overwhelming impression we have of these mutant cells, whether one looks at their spherical shape, their uniform distribution of ConA receptors under capping conditions or, more especially, their even distribution of surface-associated filamentous actin, is that they have lost their polarity at the restrictive temperature. Assuming that this is so, our observations indicate that NSF, like clathrin (Wessels et al., 2000), is required not only for some membrane uptake processes, but that its activity is essential to help establish, or maintain, the polarity of the cell. Without this, the cells could not move or cap surface receptors, whatever the mechanism of locomotion.
A migrating cell whether mammalian or amoeboid has a polarity defined by its leading front. How this external feature relates to the internal organisation of the cell is unclear; however, an internal polarity is likely to include a cytoskeleton primed in such a way that molecular components required for the advance of the leading edge are transported up to it. This polarity is, however, flexible. In the absence of any external signals, cells usually continue in the same direction for a while their movement has a persistence, which suggests that there is a positive feedback loop that gives the leading front an inherent stability. As the cell advances, the cytoskeleton is continually maintained or rebuilt so as to bring components for lamellar extension up to that front. However, many cells can change direction extremely quickly given an external cue: when this happens, a new front is established and the old one loses its activity within seconds. This means that, in the absence or presence of external cues, the leading edge and cytoskeleton communicate with one another to reinforce the existing polarity (Weiner et al., 2000).
But how can this polarity depend on NSF? In mammalian cells, the leading edge is the site at which recycling membrane is added to the cell surface (Bretscher, 1996), and this may also occur in amoebae (Aguado-Velasco and Bretscher, 1999
), although there is no direct evidence for it. We propose that the addition of this membrane to the front of the cell causes a transient and internal orienting signal to be emitted that helps organise the cytoskeleton for continued vesicle delivery in that direction. In this way, a continuation of the polarity would be achieved with the cytoskeleton itself being continuously replenished. However, this homeostasis could be over-ridden by an external hormonal signal. In this situation, the region of the cell surface most highly activated by hormonal receptors would emit a stronger internal signal, with the consequential formation of a reoriented cytoskeleton delivering vesicles to a new front. Once again, this would be stabilised by the orienting signal. If vesicle flow were stopped, this signal would cease and the cytoskeleton, and hence the cell, would lose its polarity. In this way, the maintenance of polarity would depend on a continuation of membrane recycling pathways and thus depend on NSF.
It may be that the cell polarities observed in different contexts can be subdivided into two broad classes. The first would include epithelial cell polarity where a comparatively stable arrangement exists with apical and basolateral domains of the plasma membrane separated by permanent barriers. The other class might include the polarity that exists in dividing stem cells and oocytes, or that of migrating cells or epithelial planar polarity. This latter class of polarities appears to have a more kinetic character: during the period in which they have their influence, there may be a continuing process to maintain them. Our present ignorance of the underlying molecular mechanisms that generate them means that we cannot be certain whether some, or all, depend on basically similar mechanisms. For example, it seems possible that the polarities seen in chemotaxis and planar polarity are related at a molecular level: both can be oriented by external chemicals such as a chemoattractant [fMLP or cAMP (Weiner et al., 2000)] or a morphogen [possibly a Wnt protein acting on the frizzled receptor (Adler and Lee, 2001
)]. An understanding of the basic machinery used to determine the polarity of a cell should enable one to see how those specific components used in a particular process function, and therefore what they do. Whether NSF is required to maintain all these more dynamic polarities remains to be established.
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ACKNOWLEDGMENTS |
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REFERENCES |
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