The mechanics and control of growth
The 'cell cycle' refers to the progression from the newly separated daughter to the point where it itself separates into daughters, allocating its accumulated mass and structure between them. Of course, the process depends upon the adequate functioning of the cell's resource gathering and organisation (Chapters 3 and 4) but it also requires the accomplishment of several other key assimilatory steps. The correct proteins and lipids must be formed; they have to be arranged in the relevant cytological structures and, eventually, allocated between the pro-daughter cells. Meanwhile, the entire nucleic acid complement has to have been copied, the chromosomes segregated and (at least among eukaryotes) the nuclear separation (karyokinesis) initiated. All these processes take finite and significant periods of time to complete. Throughout, their organisation and control are orchestrated by the genome, including especially the RNA of the ribosomes.
The regulation of the life-cycle events among eukaryotes is remarkably conserved, many of their features having analogues in the bacteria (Vaulot, 1995). This much is well known, the process of nuclear division having been described by nineteenth-century microscopists studying
The cell cycle. The sketch shows the vegetative growth (G) of the new cell and, at maturity, the break into mitotic division (M) and separation of the daughter cells. Completion depends crucially upon (S) the replication of the DNA which occurs only after the regulatory proteins have been 'satisfied' that the cell has all the resources necessary to sustain the daughters. Sketch based on figures in Murray and Kirschner (1991) and Vaulot (1995), and redrawn with permission from Reynolds (1997a).
organisms as mutually disparate as yeasts and frogs. As is also now well known, the primary alternation is, of course, between nuclear interphase, during which the cell increases in mass but the nucleus remains intact, and mitosis, during which the nucleus is replicated. Mitosis follows a strict sequence of steps, starting with the breakdown of the nuclear membrane (prophase); the duplicated chromosomes first align (metaphase) and then separate to the poles of the nuclear spindle (anaphase), before the propagated chromosomes become re-encapsulated in separate nuclei (telophase). The rest of the cell contents divide around the two daughter nuclei, the original maternal cytoplasm thus becoming divided to contribute the substance of each of the two new daughter cells, which are eventually excised from each other to complete the cell division. It is not yet wholly clear how elaborate organelles (such as flagella, vacuoles, eye-spots) are reallocated but the daughters soon copy what they are missing. Cell division in the markedly asymmetric dinoflagellates allocates the armoured exoskele-
tal plates between the separating daughters: each has to produce and assemble the replacement parts (for details, see Pfiester and Anderson, 1987). The cytokinetic bequest of one parental frustule requires each daughter diatom to produce anew the relevant complementary (internal) valve (see Crawford and Schmid, 1986, for more details). Similar issues of scale or coc-colith replacement respectively confront dividing synurophyceaens (Leadbeater, 1990) and coccolithophorids (de Vrind-de Jong et al., 1994).
After separation, the next generation of daughters resumes growth during the next period of nuclear interphase. Following the discovery of Howard and Pelc (1951) that DNA synthesis is discontinuous and confined to a distinct segment of the cell development, the interphase is also subdivided into corresponding the periods. These are denoted by S, signifying synthesis, G1 for the preceding gap and G2 for the succeeding gap, terminated by the inception of mitosis prophase (see Fig. 5.1).
Assembling the growing cell has been analo-gised to a factory production line, with a highly sensitive quality supervision over each process (Reynolds, 1997a). Close coordination is required to sequence the events of interphase in the correct order, to check stocks and marshal the components and, if something is missing, to determine that production should be suspended. It is also needed to initiate mitosis in a way that keeps the size of the daughter cells so evidently similar to that of the parent cell prior to its own division. The 'supervision' is, in fact, achieved through the activity of a series of regulatory proteins, knowledge of which has developed relatively recently (Murray and Hunt, 1993; good summaries appear in Murray and Kirschner, 1991; Vaulot, 1995). A maturation-promoting factor (MPF) occurs at a low concentration in newly separated daughter cells but it increases steadily throughout interphase. Purification of MPF showed it to be made of two kinds of protein. One of these, cyclin B, is one of several cyclins produced and periodically destroyed through the cell cycle, each being specific to a particular part of the cycle. Cyclin B is the one that reaches its maximum concentration at the start of mitosis. The second part of the MPF is a kinase. It had been first discovered in a strain of mutant yeast, codified cdc. The mutation concerned the presence of a kinase-encoding gene, called cdc2, and the 34-kDa kinase protein was referred to as p34cdc2. Like all kinases, it is active only when phospho-rylated (cf. Section 4.2.2). In this state, it triggers prophase spindle formation. After completion of the mitosis, the kinase is dephosphorylated and the cyclin B is rapidly degraded (by a cyclin protease), thus leading to the destruction of MPF. In the daughter cells, cyclin B is synthesised again and MPF begins to accumulate for the next division.
The cues are critical. In this instance, it is clearly the instruction to phosphorylate the kinase that triggers the mitosis. The commitment to nuclear division is, however, made earlier, when, following a sequence of signals processed through the preceding G1 period of interphase, the DNA is finally replicated (the S phase in Fig. 5.1). Activation depends upon satisfaction of resource adequacy, which is communicated by the operons informed by the intracellular-
transport and protein-synthesis pathways (see Sections 3.4.2,4.2.2). Transcription of the relevant operon genes is stimulated by the group of cAMP receptor proteins (or CRP) associated with the active pathways. Thus, marked resource under-saturation or slow delivery are reflected in weakened CRP flow and weakened operon activity. Much like the air-brakes on a train, active transport and synthesis act to suppress the cell's protective features but, as soon as normal functions begin to fail, the mechanisms for closing non-essential processes and conserving cell materials are immediately expressed. Incipient starvation and the stalling of the relevant ribosomes lead to the activation of the inhibitory nucleotides, such as ppGpp (see Section 4.3.3). These do not just arrest maturation but may induce the onset of a resting condition, with a substantial reduction in all metabolic activities, including RNA and protein synthesis and respiration. The machinery is said to remain in place for at least 200 h after such shutdowns (Mann, 1995). If renewed resources permit, however, renewed protein synthesis may be induced within minutes of their availability.
So long as the conditions remain benign, optimal functioning is supported and CRP generation is upheld. The cell recognises that it has sufficient in reserve to be able to fulfil the mitosis and so allow the DNA replication to proceed. Then, there really is no escape from the commitment. A further group of substances, called licensing factors, are bound to the chromosomes but are destroyed during DNA replication. Licensing factors cannot be re-formed while the nuclear membrane is intact, so that the DNA synthesis can occur only once per generation.
Though they lack a membrane-bound nucleus and any of the physical structure of a spindle upon which to sort the replicated chromosomes, the prokaryotes have no lesser need than eukary-otes for a closely controlled, phased cell cycle. At slow rates of growth, the DNA replication of E. coli occupies only a fraction of the generation time, giving a delineation analogous to the G1-S-G2 phasing represented in Fig. 5.1. In the fast-growing Synechococcus, DNA replication may occupy relatively more of the cell generation time. Completion of the DNA duplication is the cue for chromosome separation and cell division (Armbrust et al., 1989). Cycle regulation by analogues of MPF is probable.
All the eukaryotic species of phytoplankton that have been investigated conform to G1-S-G2 phasing (Vaulot, 1995). Curiously, the best-studied freshwater species belong to the Chlorococcales or to the Volvocales, which undergo a relatively prolonged growth period that is followed by a fairly rapid series of cell divisions, resulting in the formation of between four (as in genera of Chlorococcales such as Chlorella and Scenedesmus) to 16 or 32 (in Eudorina) or, perhaps, as many as 1000 (Volvox itself) daughter cells. Suppressed though the smooth transition between generations may be, it is also quite evident that, over two or three generations, the increase in specific biomass adheres closely to a smooth exponential rate (Reynolds and Rodgers, 1983). Reynolds' (1983b) deductions on the increase in biomass of a field population of Volvox aureus are also consistent with a smoothing of both cell growth and population growth with respect to the celldivision sequence. To treat the growth rate and the times of consecutive generations (tG) in the same terms as simple binary fission times may be cautiously justified. Based on Eq. (5.2), we deduce:
On the other hand, every confidence can be accorded to deductions about the observable rate of population growth over consecutive generations of diatoms. There is a manifest similarity of size between parent and daughter cells, owing to the shared bequest of the parental frustules. The cellular requirements of each species are also remarkably constant, at least when cell size is taken into account (Lund, 1965; see Tables 1.4, 1.5). Cells take up little more monosilicic acid than they require to sustain the skeletal demands of the imminent division (Paasche, 1980), including that needed to maintain the necessary internal concentration (see Section 4.7). These characters lend themselves to accurate computation of biomass increase from the division of cells and its direct analogy to silicon uptake (Reynolds, 1986a). Despite the complicated kinetics of silicon uptake, the constraints on intracellular transport and the intricacies of frustular morphogenesis in forming the two new frustules required to complete each cell division (see Section 4.8), the actual construction of the new silica structures is confined to a relatively short period. The latter extends from just after nuclear division to the point of eventual cell separation. Having passed G1 of interphase in its vegetative condition, the cell commences to form the new valve in a silica-deposition vesicle just beneath the plasmalemma (Drum and Pankratz, 1964). The origins of the silicon deposition vesicle and of the control of the highly species-specific patterning of the new valves are described in detail in Pickett-Heaps et al. (1990). The trigger for the process is DNA replication. Thus, no new wall forms without the initiation of the nuclear division. Equally, the commitment to division is taken before the parent cell has taken up sufficient soluble reactive silicon either to fulfil the skeletal demand of the cell division, or to be able to maintain the necessary internal concentration (Raven, 1983). This carries ecological consequences: if the requirement is pitched against low or falling external concentrations of monosilicic acid, it is possible that, in a growing population, cells begin division in a silica-replete medium but encounter deficiency before it is completed. Many cells may fail to complete the replication and die (Moed, 1973).
While external concentrations continue to satisfy uptake requirements (KU: 0.3-5 |imol L-1; Section 4.7), the demand is assuaged and the completion of the next generation can reasonably be anticipated. For all phytoplankton, the processes of gathering of raw materials equivalent to its current mass, of assembling them into species-specific proteins and lipids and, then, into the correct cytological structures and organelles, each occupy a finite period of time. Once accomplished, further time is required for the completion of the S, G2 and mitosis phases, prior to the occurrence of the final cytokinetic separation. As stated above, this event or, at least, the frequency with which it occurs, is of fundamental significance to the plankton ecologist. To predict, measure accurately or model the rate of growth of cells has long been an ambition of students of the phytoplankton. Most of the convenient, traditional determinations of the measured rates of change in numbers are, of course, surrogates of cell growth and they are always net of metabolic losses and, often, also net of mortalities of replicated cells. The rates of cell replication are rarely predictable from separate determinations of the capacities deduced from experimental measurements of photosynthetic rate or nutrient uptake rate (although these represent upper limits). It is easy to share the frustrations of all workers who have struggled with the problem of the determination of in-situ growth rates.
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