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The pulse of the machine – reevaluating tip‐growth methodology

2001; Wiley; Volume: 151; Issue: 3 Linguagem: Inglês

10.1046/j.0028-646x.2001.00243.x

1469-8137

Nicholas P. Money,

Plant Parasitism and Resistance

Behind my stepdaughter's bedroom door hangs a vertical scale that plots her height at various time intervals over the past 15 years. Extrapolation from the last three measurements suggests that she may enjoy a future career in a freak show: 'Allison, the human skyscraper.' Thankfully, I have found solace in stunning clinical reports which conclude that children do not grow at constant rates, but exhibit what clinicians refer to as 'growth spurts' (Lampl et al., 1992). What parent would have guessed this? With respect to their rates of extension then, children might seem good models for fungal hyphae and pollen tubes, because numerous studies have shown that such tip-growing cells elongate in a pulsatile fashion. But Sandra Jackson doesn't believe a word of it (Jackson, 2001; see pp. 556–560 in this issue). And now I see with eye serene, The very pulse of the machine ... William Wordsworth, She was a phantom of delight (1807) Pulsatile growth of walled cells was described first by E. S. Castle in a classic paper on the sporangiophores of Phycomyces blakesleeanus (Castle, 1940). These cells are gigantic aerial hyphae that spiral to a height of several centimeters and then form spore-filled bulbs at their tips. More recently, López-Franco et al. (1994) reported oscillations in the rate of hyphal extension in a variety of mushroom relatives and oomycetes (or stramenopile fungi: Money, 1998). The oscillatory nature of tip growth seems obvious when the position of the cell apex is measured every few seconds. Hyphae surge forward, then proceed more slowly before showing another burst of extension growth. For studies on the inhibitory effects of drugs or mutations, these subtleties are of little interest and growth rates are calculated from measurements of tip position every few minutes, or from the expansion of colonies after days of incubation. But for the investigator scrutinizing details of the growth mechanism, every second counts. Researchers studying pollen tubes also noted pulses in extension rate (Tang et al., 1992; Pierson et al., 1995). Pursuing these observations, Holdaway-Clarke et al. (1997), Messerli & Robinson (1997, 1998), and Messerli et al. (1999) showed that changes in intracellular pH and the concentration of potassium and calcium ions occurred with the same frequency as the oscillations in growth rate. In each of these studies, a short lag was measured between the growth pulses and ion fluxes, but it was unclear whether growth preceded the ionic currents or vice versa. Recently, use of an improved laser-scanning fluorescence imaging system allowed Messerli et al. (2000) to achieve higher temporal resolution of the calcium fluxes in pollen tubes: tip position was marked every 1.5 s, and calcium levels were determined every second. They confirmed the association between growth rates and calcium fluxes and concluded that the pollen tube of Lilium longiflorum rushes forward, and then a few seconds later calcium floods into the cytoplasm through channels in the plasma membrane. Finally, to complete this mini-review of the field, my laboratory measured pulses in the micronewton forces exerted by hyphal tips of the oomycete Achlya bisexualis (Fig. 1; Johns et al., 1999). The pulses in force showed the same frequency (approx. 10−2 Hz) as the excursions in growth rate characteristic of this water mold. Everyone seemed happy: calcium researchers confident in the cell biological divinity of their ion found that growth pulses fitted snugly into existing models of tip growth, and I explained with utter certainty that a cell that pulses forward is likely to exhibit precisely the kind of variations in turgor-derived force that we measured with strain gauges. As I said, everyone seemed cheerful … until now. Hyphae of the oomycete Achlya bisexualis photographed with phase contrast optics. While Sandra Jackson is especially interested in hyphal growth, her thesis relies upon features of growth rate measurements that are common to all tip-growth studies, so all readers should pay attention. In most experiments, the cells rest on a flat surface such as a coverglass, though pulses have been observed in hyphae penetrating agar medium in my laboratory (N. Money, unpublished). To measure the rate of extension, the investigator focuses the microscope objective on the cell apex. Because tip-growers are cylindrical, with hemispherical or semiellipsoidal apices, the appropriate focal plane is usually close to the middle of the cell. The position of the tip is then marked at various time intervals and rates are calculated by dividing distances traveled by time. Jackson poses the following question: what if the tip of the cell waggles up and down in the vertical axis as it extends? In principle, such a cell would appear to pulse as it undulated, even if it elongated at constant velocity. The true position of the tip is blurred by the diffraction pattern at the cell surface, and any motion in the z-axis is imperceptible to the observer if the growing cell apex does not extend much beyond the depth of the optical section (the thin slice of the cell that is viewed with a light microscope). Jackson's analysis of published data suggests that, in many cases, growth pulses might be explained by such undetectable movements in the z-axis. This is a seductive idea, but I don't believe it is strong enough to refute the diverse descriptions of pulsatile growth in tip-growing cells. The swiftest fungal hyphae and pollen tubes extend at 0.4 µm s−1. Using data from López-Franco et al. (1994), Jackson reports a positive correlation between fungal growth rates and the measured amplitude of the pulses. If z-axis motion is at the root of virtual pulses, faster-growing cells would tend to manifest greater changes in speed, so the correlation is consistent with her thesis. However, the same relationship is also anticipated for cells extending in a pulsatile fashion in a horizontal plane. An analogy may be helpful: fierce braking results in a greater change in speed during the 'Indianapolis 500' than a driver experiences in stop-and-go traffic in a city. A tip-growing cell that pursues a sinuous path restricted to the z-axis is an unlikely beast, but less confined motion in three-dimensional space is a plausible behaviour for hyphae and pollen tubes. Helical extension of hyphae and sporangiophores has been reported by many authors, and occurs in air and in solid substrates (Kaminskyj & Heath, 1992). Jackson suggests that helical growth is a potential source of artifactual measurements of growth pulses. However, the tip of a cell would have to trace a very tight gyre to appear to have a pulse rate in excess of one or two beats per minute (Trichoderma viride exhibits 14 pulses per minute; López-Franco et al., 1994). There is an even more fundamental objection to Jackson's model. While we are decades from a satisfying model that explains how a cell manages to form a cylinder when water influx tends to inflate it like a balloon, tip-growth researchers have shown recent signs of intelligence by acknowledging the mechanical intricacies of their experimental subjects. The pace at which a tip expands is limited by the rate at which vesicles arrive at the apical plasma membrane and supply the growing cell with new surface components. This exocytotic activity is affected by metabolic factors, cytoskeleton-mediated transport of vesicles, and the concentration of ions in the apical cytoplasm. The list of variables that influence the growth rate is limited only by the breadth of one's imagination. This recognition of complexity is the main message of the paper by Feijóet al. (2001) on pollen tube growth, and is a recurring theme in the symposium volume edited by Geitmann et al. (2001). Cellular growth is so complex, and involves such a medley of interacting variables, that constant extension is as unlikely as a heart that beats with the precision of an atomic clock. Do hyphae and other tip-growing cells pulse as they grow? For a cell with a slow pulse (like the hyphae of oomycetes) it may be quite straightforward to determine whether or not an apex moves in the z-axis by using confocal microscopy to reconstruct images of cell shape before, during, and after a perceived growth pulse. Until such experiments have been performed, arguments about the strengths and weaknesses of different published studies can be anticipated, but when the smoke clears I suspect that pulsatile growth will be recognized as a universal feature of polarized cellular development. Whatever the outcome, Sandra Jackson has performed a very useful service by making all tip-growth researchers reevaluate their methodology. She should receive some interesting mail.