Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

Abstract

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two-to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate.Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.In their pioneering work on glycosaminoglycan (GAG) ~ synthesis by cultured cells, Kraemer and Tobey (14) showed that a significant fraction of trypsin-releasable (i.e., surface) GAGs was shed during mitosis. Although the Chinese hamster ovary (CHO) cells analyzed were grown in suspension, viewed from the perspective of recent studies of cell adhesion (e.g., reference 32), it seemed likely that the shedding of GAGs was related to the familiar ease with which mitotic cells are detached from monolayers. We hypothesized that any effect of shedding would be reinforced by a coordinate reduction in GAG synthesis that would delay replacement of surface GAGs, and that a rapid resumption of GAG synthesis in early G i would be required for cell reattachment.A parallel motivation for our studies arose from the accumulating evidence that membrane vesicle-mediated transport is depressed during mitosis. This is shown clearly in work from our laboratory demonstrating depression of endocytosis ( 1, 2) and transferrin recycling (35) and from that of Warren and colleagues showing a decrease in surface transferrin receptors (41), and in depression of histamine secretion (9). These studies focus primarily on fusion and budding events at the plasma membrane. In addition, the insertion of the G t Abbreviations used in this paper. CHO, Chinese hamster ovary; GAG, glycosaminoglycan.protein of vesicular stomatitis virus is also depressed during mitosis (42), which suggests a defect in its processing through the Golgi apparatus. Since carbohydrate chains of GAGs are largely assembled within the Golgi apparatus by multiple enzymes (31), a general defect in processing should affect GAG synthesis. Moreover, the apparent localization of sulfation to the trans-Gol~ apparatus (7, 44) would serve to focus on later steps of the processing sequence.We describe here the rates of labeled sulfate incorporation into CHO cells during mitosis, in their subsequent movement into G1, and in late G2. We followed incorporat...

In Vitro Synthesis of Sulfated Glycosaminoglycans Coupled to Inter-compartmental Golgi Transport

“…Just 1 h after nocodazole removal the cells recovered their full GAG synthesizing capacity. At this stage, when most cells were entering early G 1 , we saw an overshoot of GAGs synthesis very similar to the one reported in the autoradiographic study of Preston et al (55).…”

Membrane traffic between secretory compartments is differentially affected during mitosis.

“…previously reported (Preston et al, 1985); thus, the 3% contamination with interphase cells could give rise to secretion of no more than 9-120/o of GAG chains synthesized. Because we observed secretion of more than 85%/o, we conclude that the majority of GAG chains secreted is from the mitotic pool.…”

Regulation of Ca2+ influx during mitosis: Ca2+ influx and depletion of intracellular Ca2+ stores are coupled in interphase but not mitosis.

“…However, unlike interphase cells, in mitotic cells the elevation of [Ca2"], is not sustained and "5Ca2" influx is not stim-Introduction A wide range of membrane properties is altered during mitosis. This includes endocytosis (Berlin et aL, 1978), exocytosis (Hesketh et al, 1984), receptor recycling (Sager et al, 1984;Warren et al, 1985), glycosaminoglycan secretion (Preston et al, 1985), glycoprotein processing (Warren et al, 1983), and, we have shown recently, membrane signal transduction (Volpi and Berlin, 1988). In interphase HeLa cells, activation of the histamine Hi receptor leads to an elevation of intracellular free calcium ([Ca2+]D).…”