Periodic synthesis of microtubular proteins in the cell cycle of Physarum.

Abstract

Periodic polypeptide labeling over the naturally synchronous nuclear replication cycle of Physarum polycephalum was analyzed by fluorography of two-dimensional electropherograms. Two sets of polypeptides, denoted as P and Q, showed strong periodicity; they were maximally labeled just prior to mitosis. This periodicity was shown to reflect synthesis rather than turnover or recovery. Both P and Q copolymerized with porcine microtubular proteins and displayed electrophoretic properties similar to those of porcine tubulins. The significance of the periodic synthesis of these microtubular proteins is discussed as a possible component within the chain ofevents that establishes the high mitotic synchrony of Physarum syncytia. The replicative cell cycle of eukaryotes is marked by major cellular events such as entry into DNA synthesis (S phase) and progression through the stages of mitosis. Do these cellular events reflect molecular periodicities in protein synthesis?One can distinguish between division cycles that generate apparently equivalent daughter cells and those cycles that yield frankly distinct progeny. In the differentiative cell cycle ofCaulobacter crescentus, the generation of a new cell type, the swarmer, is accompanied by strong periodicities in the synthesis ofa number ofpolypeptides such as the flagellin needed for this new cell type (1).The situation for proliferative systems with equational division is less clear. Experimental analysis in these systems is complicated by the problem of distinguishing between a natural, endogenous periodicity and an artifactual one induced by the synchronization procedure (e.g., see ref.2). Two approaches have arisen to deal with this problem: (i) postsynchronization methods such as elutriation or mitotic detachment, to separate prelabeled cells into temporal classes; and (ii) natural synchrony in a syncytium large enough for biochemical analysis, such as that of the myxomycete Physarum polycephalum (see ref. 3).The first approach has been used to analyze the periodicity of labeling of polypeptides in Saccharomyces cerevisiae (4) and HeLa cells (5). Among the polypeptides that can be identified in two-dimensional electropherograms from Saccharomyces or from HeLa, the amplitude oflabeling never changes more than 3-fold.We have adopted the second approach, using Physarum. The extreme natural synchrony might permit detection of periodicities that would be obscured by a loss of resolution in postsynchronization methods.We report that Physarum polypeptides with properties of microtubular proteins are synthesized preferentially in the premitotic interval of the division cycle. The amplitude of the periodicity is at least 30-fold. MATERIALS AND METHODSCultures. The general culture methods have been described by Mittermayer et al. (6). Microplasmodia of strain CL (7) were grown in suspension culture in a simplified soy medium (SSM) adapted from a medium of E. Brewer and B. Prior (personal communication): per liter, 10 g of Difco BactoSoytone, 3 g of yeast extract, 9 g ...

Cell cycle stage-specific accumulation of mRNAs encoding tubulin and other polypeptides in Chlamydomonas.

“…5)], we cannot unambiguously correlate the period of tubulin synthesis with either mitotic apparatus construction or flagellar regeneration. However, similar cell cycle changes in tubulin synthesis in Physarum syncitia (11) and HeLa cells (3), two cell types that do not have flagella, argue that there may be other requirements for tubulin synthesis during the cell cycle, perhaps in construction of mitotic apparatus.…”

“…In Physarum, polypeptides identified as tubulins increase in relative rate of synthesis about 30-fold just prior to mitosis (11). Tubulins are among the few polypeptides shown to vary a few fold in relative rate of synthesis during the HeLa cell cycle (3).…”

Patterns of tubulin isotype synthesis and usage during mitotic spindle morphogenesis in Physarum

“…Thus, the plasmodium provides a unique opportunity for the study of the molecular biochemistry of mitosis, as the polymerization of tubulin in the plasmodial nuclei of Physurum is a very specific event marker for the initiation of spindle biogenesis. In addition, the coordinate synthesis of tubulins occurs in the 2-3-hr period of late G2 immediately preceding mitosis [Chang et al, 1983;Laffler et al, 1981;Schedl et al, 19841.…”

Photomorphogenesis in Physarum: induction of tubulins and sporulation-specific proteins and of their mRNAs.

“…(26). These investigators have presented strong evidence that the syntheses of the a1 and P32 tubulin variants increases in the late G2 phase of the cell cycle and precedes the appearance of microtubules in the mitotic spindle apparatus (27). Therefore an alternative function of T1 and T2 during fruiting-body formation could be their participation in the presporangial mitosis (11).…”