Tubulin synthesis in a temperature-sensitive mutant of Chlamydomonas reinhardii

TUBULIN

SYNTHESIS

MUTANT

IN A TEMPERATURE-SENSITIVE

OF CHLAMYDUMOIVAS

REfNHARDfl

SUMMARY A temperature-sensitive mutant (TSF-I) of Cl~lomvdomo~tcrs reinhardii which exhibits altered regulation of tubulin synthesis has been isolated. This mutant grows equally well at permissive (25°C) and non-permissive (36°C) temperatures but possesses tlarclla only at 25°C. As with wild-type cells, w,hen flagella are detached by ‘pH shock’ at 25°C there is a rapid regeneration of flagella and a marked induction of tubulin synthesis, the major flagellar protein. However, if flagella are removed at 25°C and the cells immediately placed at 36°C there is little or no flagellar regeneration or tubulin induction. If these flagella-less cells are maintained at 36°C and subsequently shifted back to 25”C, there is a rapid initiation of both flagellar outgrowth and tubulin synthesis. An additional temperature-sensitive phenotype exhibited by TSF-I when shifted from 25 to 36’C is a spontaneous detachment of flagella. Associated with the loss of tlagella is limited (but perhaps repeated) flagellar regeneration and a marked increase in tubulin synthesis. Interestingly, ‘pH shock’ treatment at 30 or 60 min after the shift to 36°C results in a rapid de-induction of tubulin synthesis. This complements the observation that flagellar excision by ‘pH shock’ just prior to a shift to 36°C results in little or no tubulin induction. Taken together these results suggest that two independent pathways for tubulin induction may be operable in TSF-I. The short response times observed in both the shift-up and shift-down experiments demonstrate that the conditional process involved responds very rapidly to both positive and negative temperature changes and, moreover, indicate that this process may be intimately associated with the regulation of both tlagellar regeneration and flagellar tubulin synthesis.

Tubulin induction in Chlamydomonas reinhardii has been used by our laboratory as a model system for exploring mechanisms of gene regulation in a eukaryotic organism. Studies have focused on the control of biosynthetic events associated with tlagellar regeneration in this unicellular, biflagellate alga. The most striking response of cells to flagellar detachment is a rapid and extensive induction of the synthesis of tubulin, the major structural protein of the flagellum. Within 10 min after the loss of flagella, tubulin mRNA is present and actively translated in polyribosomes. Peak tubulin production (i.e. approx. 15% of total protein synthesis) is achieved within

30 min and is maintained at high levels for nearly 60 min. After flagellar regeneration has ceased, and as restoration of cytoplasmic reserves of flagellar components is being completed, the synthesis of tubulin sharply declines until little or no tubulin synthesis can be detected at 3-31 h after flagellar excision. A question of prime importance to the control of the entire cycle of tubulin induction and de-induction relates to the nature of the intracellular signals involved in triggering and maintaining the induction. We ’ Present address: Drexel University. Department ot Biological Sciences. Philadelphia. PA 19104, ISA.

330

Gealt and Weeks

have taken two long-term approaches to examining the signalling mechanisms. The first involves the use of various chemical reagents which inhibit or modify the process(es) of flagellar regeneration and/or tubulin induction. To date several reagents have been found which block flagellar regeneration but do not prevent the induction of tubulin synthesis [l]. Additionally, one chemical, amiprophos methyl (APM), was discovered not only to affect flagellar outgrowth but also to inhibit selectively the induction of tubulin synthesis [2]. The second major approach is aimed at isolating temperature-sensitive mutants of Chlamydomonas reinhardii with altered regulation of tubulin synthesis. The long-term goal of such efforts is to provide cells with conditional defects both in key pathways involved in the initiation and control of signal production and in the cellular machinery involved in receiving and responding to the intracellular signal(s). In this report we describe the major phenotypic and biochemical characteristics of a temperature-sensitive mutant which exhibits altered regulation of tubulin synthesis at non-permissive temperatures. This mutant is also shown to possess flagella which are exceptionally vulnerable to spontaneous detachment at elevated temperatures. A number of quite unique responses of these cells to flagellar loss at non-permissive temperatures are examined.

METHODS Growth and media Chlumydomotws reinhurdii 137~ NO+ (from U. Goodenough) cultures were maintained on a 12 h light: 12 h dark cycle in modified Sager & Granick medium 2 [3] with 0.3% sodium acetate as described previously [4]. Gametes were obtained by overnight nitrogen starvation of vegetative cells in nitrogenfree and sulfate-free medium V [I]. Tris-acetate phos-

137 c NO mt*

137cNOml-

-N uv

-N

I 137 c NO ml+

I 137 c NOml-

gome,es

double

detlaqellation 01 35’

glutaraldehyde treatmen

7 137 c NO mt + gomefes

Mating

I37

I

Reaction

t c NO mtI

“corpses”

(at 35O)

filter ihrough polypropylene 137~

g~meles

NO ml+gomeles

filter

(unable

lo male1

plate on TAP-agar incubate at 35’

I Select

colonies

with altered

morphology

1. Flow diagram of the isolation protocol used to enrich for temperature-sensitive flagellar regeneration mutants of Chlamydomonas reinhardii.

Fig.

phate (TAP) medium [5] was used to grow and maintain cultures during the mutant isolation protocols. Induction of gamete formation in cells used for mutagenesis was performed by resuspending cells in TAP medium without nitrogen (TAP minus N). Cultures at the (permissive) temperature of 25°C were grown on a New Brunswick Scientific gyrotory platform shaker (either model G2 or GIO-21) at IOOI50 rpm. Temperature shifts to 36°C (the restrictive temperature) were accomplished by incubating a small volume of cells (3-30 mlj in prewarmed vessels in a New Brunswick Scientific G-76 water bath shaker.

Mutant

isolation

Our aim was to select a conditional mutant strain unable to regenerate flagella under non-permissive conditions. The mutant isolation scheme is illustrated in fig. I. Wild-type cells (C. reinhurdii 137~ NO mf ‘.) were grown in TAP. The cells were transferred to TAP minus N to induce gamete formation. then mutagenized with ultraviolet light for 180 set (8% survival) and, finally, incubated overnight in the dark at 25°C to allow gametogenesis to proceed. The next morning the cells were deflagellated by the ‘pH shock method’. i.e. the nH was lowered to 4.7 with acetic acid (I N) for 15-90 set after which the medium was neutralized with KOH (I N) 161. These cells were then incubated at 35°C to allow fl&&llar outgrowth in wildtype cells. A second deflagellation and incubation at 35°C was performed on the presumption that cells which were unable to make flagellar proteins might still be able to regcneratc flagella after the first de-

Tuhirlin flagellation by utilizing intracellular pools of flagellar proteins [4, 71. Presumably regeneration after two successive flagellar detachments would require new flagellar protein synthesis by the cells. Since only cells which were phenotypically wild-type for flagellar protein synthesis and assembly would be capable of regenerating flagella, it would be only these gametes which would be capable of mating. The mutagenized, double-deflagellated mt+ cells-were mated for 10 min at 35°C with 137~ NO mt cells which had been fixed with glutaraldehyde (I %,) and subsequently washed with, and resuspended in, sterile water. These mt- cells, which are termed ‘corpses’ 18. 9] retain their competence for mating pair formation. The mating pairs (or clumps) are quite stable for several hours. The mated oairs were removed bv filtration through a stack of f;ve polypropylene filters (Gelman. IO urn pore size) flO1. The non-matine cells were then piated on TAP-agar plates (either ?o.S or 2.5% agar) and incubated at 35°C. Colonies which exhibited an altered morphology (clumped, grainy appearance) with respect to wild-type (dispersed, shiny appearance) were picked and grown in test tube cultures to check for swimming capability and the presence of flagella at the permissive and non-permissive temperatures. Since Chiamydomona.s naturally resorbs its flagella during the cell cvcle prior to cell division, and-then regenerates flagella following cytokinesis, the lack of flagella following overniaht growth at 35°C was taken ai evidence fo; the inability to regenerate flagella. Thus, cells were selected which possessed the ability to produce flagella at permissive temperatures but not at non-permissive temperatures. Since the construction of flagella involves the interdependent functioning of many different proteins, it is obvious that many cells of the selected phenotype would not be altered in their regulation of tubulin synthesis. That is, in some cells tubulin synthesis would be induced by deflagellation whether or not the cell was capable of regenerating flagella. Therefore, cells originally selected as conditionally altered in flagellar regeneration were specifically screened for alterations in the regulation of tubulin synthesis by examining the spectrum of proteins produced following deflagellation; the requirement being that cells deflagellated under non-permissive conditions do not synthesize tubulin. Our criteria also included the requirement that the cell be capable of growth at the non-permissive temperature. In this way we could be certain that the cells were capable of synthesizing tubulin for mitosis and other physiological processes requiring tubulin synthesis and microtubule assembly.

Labelling

und gel electrophoresis

Cells were pulse-labelled for various periods of time with [?S]H,SO, as described previously [ 11. Following labelling, 10 vol of acetone were added directly to the incubation mixture. This precipitated proteins both within the cell and those released into the medium. Dried acetone powders were dissolved in 2% sodium dodecyl sulfate (SDS), 5% /3-mercaptoethanol, 10% glycerol and 60 mM Tris-HCI (pH 7.6) and an aliquot containing 2.5~ 1W to IX 106 cpm was electrophoresed

synthesis nzutunl

331

on 8% SDS-polyacrylamide gels as described previously [4]. Autoradiographic exposure of the dried gel was 12-60 h with Kodak XRS film.

Cell body isolation In order to separate cell bodies from detached flagella, cells in suspension were underlaid with ?S% sucrose and centrifugated at 1800 t-pm in an IEC Model CV. During th:s centrifugation,‘cell bodies pellet on the bottom of the tube. Detached flagella dc not penetrate the sucrose layer. Cell bodies and fla gella were precipitated separately with acetone.

Cells were examined by light microscopy using phase contrast optics. The samples were prepared by adding a cell suspension to an equal volume of 0.15 M NaCI, 0.1 ‘ii. sodium azide solution, followed by the addition of 2 vol of 2% glutaraldehyde, 50 mM K,HPG,/KH,PG, buffer (pH 6.8). In order to examine the point of flagellar breakage in TSF-I at non-permissive temperatures, low levels (5 @M) of amiprophos methyl (APM) 123 were added to block flagellar elongation. Samples for scanning electron microscopy (SEM) were fixed in 2 % glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.0) for a period of l-3 h. Cells were pelleted and resuspended in 0.1 M sodium cacodylate buffer (pH 7.3). Samples were then critical point-dried on glass platelets and shadowed with platinum : palladium in a Denton Vacuum DV-502 at an angle of 45”. Electron microgrdphs were taken on an ETEC Autoscan Electron Microscope at 20 kV.

RESULTS The initial protocol used in our laboratory for the selection of TSF-I and other cells with temperature-sensitive defects in flagellar regeneration and in the regulation of tubulin synthesis called for the isolation of cells that could not participate in the mating reaction between gametes of opposite mating types by virtue of their lack of flagella at non-permissive temperatures (5 36°C). Those cells that did not participate in pairing but were able to swim when returned to permissive (25°C) conditions were selected for further analysis. TSF-I was the first such variant to be selected for detailed phenotypic characterization. TSF-1 was found to possess flagella wher, grown at the permissive temperature (25°C)

332

Dealt und Weeks

Time

(min)

Fig. 2. Loss of flagella by Chlumydomonas reinhardii TSF-I following a temperature shift from 25 to 36’C. The percentage of cells retaining 0, 2; 0. 1, or X, nd flagella- is plotted as a function of time after the shift to 36°C. Each time point represents the average of 3-5 experiments with-at least-100 cells counted per experiment.

but to lack them when grown at the restrictive temperature (35 or 36°C). Growth rates were similar at both temperatures. At 25°C approx. 90 % of the cells possessed two flagella (this percentage was indistinguishable from the 137~ NO parental strain). A maximum of 2-3s of the TSF-1 cells appeared to have only a single flagellum. Unexpectedly, when gametic cells were shifted from 25 to 36”C, flagella spontaneously detached from the cell bodies (fig. 2). The detached flagella were found floating ‘free’ in the medium. The percentage of the population retaining both flagella attached to the cell body dropped precipitously at 36°C. By 30 min after the temperature shift, only 50% of the cells possessed two flagella. By 120 min this figure dropped to 5 %. Importantly, the number of cells in the population with only a single flagellum increased during the first 30 min of incubation at 36°C. The observation of singly flagellated cells suggested that flaExp

Cell

Rrs

127 (IWO)

gella were being lost one at a time from the cell body. It should be noted that the wildtype parent does not spontaneously lose flagella when incubated at 36°C. To confirm the light microscope observations, a SEM examination was made of cells undergoing flagellar loss (fig. 3). In the presence of APM (amiprophos methyl), a reagent which prevents flagellar regeneration in C. reinhardii [2], we observed cells possessing two flagella (fig. 3~); one flagellum (fig. 3h); and no flagella (fig. 3a). The cell wall collar through which the flagellum normally extends remains as an obvious marker of the lost appendages (fig. 3a, h, cf, arrows). Fig. 3c, d indicates that, when cells are incubated at 36°C even in the absence of APM, the cell population consisted of cells with two, one, or no flagella. For example, in fig. 3b, d it is possible to see clearly the empty cell wall collar on cells which have lost one flagellum but still retain their other flagellum. The observation of cells with a single flagellum indicates again that flagella were lost independently. The observation of cells with empty cell wall collars, coupled with our light microscope observation of free floating flagella suggests that the cell loses an entire flagellum as a unit and that breakage is not at random along the length of the flagellum. Tub&n induction When wild-type C. reinhardii is deflagellated, the cell responds both by regenerating new flagella and by inducing the synthesis of tubulin [l, 41 and other flagellar proteins [ 111. As shown in fig. 4, A-E, following deflagellation of 137~ NO mt+, the combined synthesis of a- and /I-tubulin accounts for approx. 20% of the total protein being synthesized. When TSF-1 is deflagellated at the permissive temperature it also reflagellates and, as indicated in fig. 4,

Fig. 3. SEM of C‘ldum.whmonas rcithrtlii ‘I‘SF-I following incubation at 36°C. Cells were incubated for (A) 60 min at 36°C in the presence of IO-” M APM; (R) IS min in the presence of .4PM; (C) 45 min

without any drug present; (I>) 30 min without any drug present. Arrows indicate presumptive flagcllar insertion sites. Bar. 2 pm.

F-J, responds by inducing the synthesis of tubulin. It may be noted that there is a low basal level of tubulin synthesis in the non-deflagellated control cells of both the 137~ NO (wild-type) and the TSF-I strains. Tubulin induction appears somewhat less intense in TSF-I than in the wild-type, but the level of tubulin synthesized is, nonetheless, many times greater than the nondeflagellated basal level. The duration of enhanced tubulin synthesis was comparable in wild-type and TSF-I.

When TSF-I is shifted to 36°C. its flagella detach spontaneously (see above) and there is an accompanying induction of tubulin synthesis (fig. 5). Tubulin synthesis is initiated within 15 min after the temperature shift and the level of synthesis remains elevated for approx. 180 min. (The duration of tubulin synthesis is variable, generally decreasing to control levels by 180 min but sometimes continuing beyond 180 min.) Also to be noted is the fact that in both wild-type and TSF-I a number of high

334

Gealt and Weeks A

15-

45-

105-

165-

4560

IOS120

o-

Fig. 4. Flagellar tubulin induction following deflagellation at the permissive temperature (25°C). A-E, Results using the parental wild-type 137~ NO mr-; F-J, results with mutant strain TSF-I. Controls (A, F) are the results of labelling non-deflagellated cells.

Exp

Cell

Res

127 (1980)

G

E

165180

15

Fig. 6. SEM of C. reinhardii cubation at 36°C: showing (A)

B

TSF-I following inshort length flagella:

IS- 45-

105- zcis30 60 120 180 Ken- deflogellated 360

Fig. 5. Induction of tubulin as a result of incubation of TSF-I at 36°C. Mutant strain C. winhardii TSF-I was shifted to 36°C and pulse-labelled for the time periods indicated after the shift. These cells were not deflagellated but did undergo spontaneous flagellar loss.

(B) unequal length flagella. (A, B) 15 min at 36°C. Bar, I pm.

molecular weight proteins show a marked increase in synthesis following the temperature shift to 36°C. Since TSF-I responds to the spontaneous loss of flagella at the restrictive temperature by inducing flagellar protein synthesis, we postulated that the cell was, indeed, attempting to retlagellate and that the newly formed flagella were continuing to detach spontaneously from the cell. If that were the case, it followed that after a shift to 36°C some cells should possess shorter than normal length flagella or two flagella of different lengths. When samples of TSF-1 incubated at 36°C are examined by SEM it is possible to find consistently a small number of cells which contain variant length flagella (fig. 6). These cells apparently begin flagellar regrowth and thus possess shorter than normal length flagella. With additional time at the restrictive temperature even these flagellar stubs are shed and, as indicated in fig. 2, after 180 min of incubation at 36°C more than 95% of the cells are completely without flagella. From this one would predict that all tubulin synthesized by these cells during the incubation at 36°C should be assembled into microtubules in flagella which are later shed from the cell. In fig. 7 we compare a sample of TSF-1 (cells plus proteins in the surrounding medium) labelled for the period 75-105 min after the shift to 36°C with the purified cell bodies labelled for the entire 105 min following the temperature shift. The results clearly indicate that none of the tubulin synthesized after the shift to 36°C is retained by the cell. That is, comparison of the newly synthesized proteins of cells removed from their culture medium by centrifugation through 25% sucrose (fig. 7B) with the proteins present in cell bodies plus the external medium, reveals that most, if not all, of the newly produced tubulin is

Fi,q. 7. Comparison of tubulin present in acetone precipiration of an entire iabelling preparation and isolated cell bodies of TSF- 1. Cells in A were labellcd 75-105 min after being shifted to 36°C; the entire reaction mixture was precipitated with IO vol of acetone prior to gel electrophoresis. Cells in R were labelled for I05 min (O-105 min after temperature shift) then pelleted through 25 % sucrose to separate cell bodies from Flagella. The cell bodies were rcsuspended in 0.5 ml H,O, and precipitated with 10 vol of acetone prior to gel eiectrophoresis.

present in the medium, i.e., the tubulin is being lost from the cell as partially regenerated flagella or flagellar components are shed from the cell. As shown above, tubulin synthesis is induced in TSF-I when flagella are detached by ‘pH shock’ (see Methods) at 25°C or when spontaneous flagellar loss occurs at 36°C. The distinguishing characteristic of TSF-I is, however, that when the cells are first deflagellated at 25°C and then shifted immediately to 36”C, there is little or no induction of tubulin synthesis. This is shown in fig. 8 where induction in the absence of flagellar detachment upon shift to 36°C (fig. 8D-F) is compared with the lack of tubulin induction in cells deflagellated just prior to the shift to 36°C (fig. SG, H). This lack of induction also contrasts sharply with the marked stimulation of tubulin

336

Gealt and Weeks

Fig. 8. Comparison of tubulin induction response by C. reinhardii TSF-I to flagellar loss by excision at 25 and 36°C or spontaneous fall-off at 36°C. A, Labelling for I5 min of non-deflagellatedcells at 25°C; II, C, response of incubation at 25°C following deflagellation; D-F. induction bv incubation at 36°C without deflagellation; G-L, response at 36°C following deflagellation with no pre-incubation at 36°C (G, H), 30 min pre-incubation at 36T (I, .I), and 60 min preincubation at 36°C (K, L). All labelling periods are 15 min beginning at the time followinr flanellar detachment or temperature shift indicated below each track.

production in cells from the same deflagellation mixture maintained at the initial temperature of 25°C (fig. 8G, H vs B, C). In addition, it can be seen that if cells that have undergone induction due to spontaneous flagellar loss at 36°C (i.e. tracks D-F) are exposed to ‘pH shock’ at 30 min after the shift to 36°C there is a marked ‘de-

induction’ of tubulin synthesis (compare fig. 81, J with D-F). Similar results are obtained with cells incubated for 60 min prior to the pH shock treatment (fig. 8K, L). Fig. 9 presents an expanded time course of the induction of tubulin synthesis which occurs after a shift to TSF-1 to 36°C (fig. 9B-F) and the corresponding lack of induc-

9. Tubulin induction at 36°C by C. reinhardii TSF-I without or v&h flagellar detachment prior to temnerature shift and tubulin induction-in cells shifted back to 25°C. A, H, Result of labelling non-deflagellated cells at 25°C; B-I-‘, result of shifting to 36°C and allowing flagellar fall-off; G-J, results of deflagellating cells before shifting to 36°C (no ire-incubation); K, Ly results of returning cells to 25°C after 180 min incubation at 36°C following deflagellation. All labelling periods are 15 min. B-L, The labelling period following temperature shift is indicated below each track. Fig.

Err, Cell Rrs 127 (IWO)

Tubulin synthesis

tion when cells are deflagellated just prior to exposure to 36°C (fig. 9G-J). In addition, fig. 9K, L demonstrates the temperaturesensitive nature of tubulin synthesis in TSF-I. In this experiment, an aliquot of the cells whose flagella were removed just prior to the shift to 36°C (i.e., cells used for the experiment depicted in fig. 9G-.I) was maintained for 180 min at 36°C before being returned to 25°C. Within 15-30 min after the shift back to permissive temperatures tubulin synthesis could be detected (fig. 9K). By 45-60 min (fig. 9L) tubulin induction was quite marked. Concomitant with the induction of tubulin synthesis is the initiation of flagellar regeneration (data not shown). In separate experiments (not shown), cells maintained overnight at 36”C, and which were therefore without flagella, showed a similar rapid response (i.e., tubulin induction and flagellar regeneration) when shifted to permissive temperatures. Thus, while TSF-I deflagellated at 25°C and maintained at 36°C may lack the ability to regenerate flagella and initiate tubulin synthesis, the cellular mechanisms involved in both processes quickly return to their normal responsive state upon restoration of favorable temperature conditions.

DISCUSSION The use of genetically altered variants has, historically, provided a powerful tool for probing the regulation of physiological processes. Mutants of Chlamydomonas with altered flagellar structure have been isolated [12-151 and correlative studies have shown the structure-function relationship of certain flagellar proteins [16]. Recent reports have indicated the feasibility of isolating conditional mutants in various flagellar functions [17, 181. These results pointed

mutant

337

to the feasibility of selecting temperaturesensitive mutants of Chlamydomonas with altered regulation of the tubulin induction that accompanies flagellar regeneration. Various screening schemes were devised to select for cells with altered ability to regenerate flagella at non-permissive temperatures, but which showed a normal pattern of flagellar regeneration at permissive temperatures. The rationale was that some cells selected by such a screen might show an impaired or modified ability to produce tubulin and/or other flagellar protein under the non-permissive conditions. One screening technique based on the inability of flagella-less cells to participate in the flagellardependent mating reaction (see Methods) yielded a number of variant cells which possessed flagella when grown at 25°C but were devoid of flagella when grown at 35°C. TSF-1 was initially chosen as one cell in a class of variants that showed rapid regeneration of flagella and concomitant induction of tubulin synthesis upon shift from 36 to 25°C (fig. 9). Analysis of TSF-I has shown that it not only possesses a desirable temperature-sensitive phenotype (i.e., a marked alteration in the regulation of tubulin synthesis and flagellar regeneration at 36°C) but also has a number of additional and unusual temperature-sensitive traits. In describing the TSF-1 phenotype, mention should first be made of the fact that TSF-1 cells grow and divide in an apparently normal manner at both 25 and 36°C. Thus the lesion that prevents flagellar tubulin production at 36°C has no apparent influence on the synthesis of tubulin required for spindle formation, cytoskeletal assembly, and basal body duplication. In addition, flagellar regeneration and, as shown in figs 4 and 8. tubulin induction at 2s”C is similar to tha! occurring in parental wild-type cells. (Other Exp Ceil

Res

127 (/PROi

338

Gralt and Weeks

flagellar proteins are likewise induced as a result of flagellar detachment (see [ 1 I]) but discussion here is focused exclusively on tubulin synthesis.) Only when subjected to 35°C are deflagellated cells unable to regenerate flagella or support the induction of tubulin synthesis. The spontaneous loss of flagella from TSF-I that occurs with a shift of temperature from 25 to 36°C (fig. .2) has associated with it a number of unusual and unexpected properties in regard to the control of flagellar regeneration and tubulin induction. The fragile flagella phenotype, per se, is rare among the mutants we have thus far examined, but has been observed in a number of temperature-sensitive mutants isolated and characterized by others interested in flagellar structure and function [ 181. In the case of TSF-1, flagella are shed at a relatively rapid rate and on an apparent one at a time basis. Breakage of the flagellum appears to be near its base and within the cell wall collar or channel (fig. 3). Limited regeneration of flagella may occur following spontaneous detachment since short flagella are observed a short time after flagellar shedding begins (fig. 6). (No short flagella are observed if cells are shifted to 36°C in the presence of inhibitors of flagellar outgrowth (fig. 2), indicating again that breakage occurs near the base rather than along the entire length of the flagellum.) Associated with this limited, but perhaps repeated, attempt at flagellar regeneration is a striking increase in tubulin synthesis. In marked contrast, if flagella are detached from TSF-I by pH shock just prior to the shift to 36”C, there is, at best, a minor and short-lived induction of tubulin synthesis (figs 8, 9) with little or no flagellar regeneration (data not shown). Moreover, if cells already producing increased amounts of tubulin at 30 or 60 min after the shift to 36°C are subjected to pH

shock there is a rapid de-induction of tubulin synthesis. Why does spontaneous detachment of flagella at 36°C lead to an induction of tubulin synthesis while removal of flagella by the pH shock method at 36°C does not? One possibility is that the lack of flagellar regeneration after the pH shock treatment is coupled with a lack of tubulin induction (as in the case of wild-type cells treated with APM prior to flagellar detachment [2]), whereas the flagellar outgrowth associated with spontaneous flagellar shedding may, through a separate mechanism, trigger tubulin production. It is possible, for example, that since a cell may shed one flagellum‘at a time, that the resorption of the remaining flagellum may drive the partial regeneration of a new flagellum [ 11, 191 and this may: in turn, trigger the production of tubulin and other flagellar proteins. It must be borne in mind, however, that earlier studies have shown that in wild-type cells, deflagellation (by pH shock), per se, without flagellar regeneration is sufficient to cause the induction of tubulin synthesis [l, 1 I]. Additional studies will be required to determine if there are normally separate mechanisms which the cell can use to initiate the synthesis of flagellar proteins or if the alterations in tubulin synthesis observed in TSF-1 are unique to this particular mutant. In any event, the lack of flagellar tubulin production in TSF-I grown at 36°C and its immediate induction upon shift to 25°C indicates that one or more key reactions needed to signal for the production and/or translation of tubulin mRNA are defective in TSF-1 at non-permissive temperatures. The availability of this and other such mutants with conditional defects in the production of tubulin and other flagellar proteins may prove crucial to elucidating the mechanisms involved in the

Tuhulin

regulation of protein gellar regeneration.

synthesis during

fla-

We wish to thank Dr Manfred Baver for his exnert guidance in the use of the SEM.. This research is supported by grants from NSF (PCM-7702930), NIH (CA-06927, RR-05539), and an appropriation from the Commonwealth of Pennsylvania.

REFERENCES 1. Weeks. D P, Collis, P S & Gealt, M A. Nature 268 (1977) 667. 2. Collis, P S & Weeks, D P, Science 202 (1978) 440. 3. Sager, R & Granick, S, Ann NY acad sci 56 (1953) 831. 4. Weeks, D P & Collis, P S, Cell Y (1976) 15. 5. Amrhein, N & Filner, P, Proc natl acad sci 70 (1973) 1OY9. 6. Witman. G B, Carlson. V, Berliner, J & Rosenhaum, J L, J cell biol54 (1972) 507. 7. Rosenbaum, J L. Moulder, J E & Ringo, D L. J cell biol 41 (1969) 600. 8. Goodenough, U W & Weiss, R L, J cell biol 67 (197.5) 623.

synthesis

mutant

339

9. Goodenough, U W & Forrest, C L, The molecular basis of ceil-cell interaction (ed K A Lerner ct D Bergsma) pp. 429438. Alan R. Liss. New York (1978). IO. Forest. C L & Togasaki, R K, Proc natl acad sci US 72 (197.5) 3652. II. I.efehvre, P .4, Nordstrom, S A, Moulder. J I+ & Rosenbaum. J L, J cell biol 78 (1978) 8. 12. Lewin, R, J gen microb 1I (3954) 358. 13. Randall, J, Cavalier-Smith. T, McVittie, A. War:. J W & Hopkins, J M. Dev biol. suppl. I (1967) 43. 14. Randall. J & Starling, D. The genetics of algae (ed R A Lewin) pp. 49-62. University of Califomta Press, Berkeley and Los Angeles, CA (1976). IS. Warr, J, McVittie. A, Randall, J & Hopkins, J, Genet res 7 (1966) 335. 16. Witman. G B. Fay, R & Plummer. J. 1 cell brol 76 ( 1978) 72Y. 17. Jarvik, J, Lefebvre, P A & Rosenbaum. J L. J cell biol70 (1976) 149a. 18. Huang, B. Ritkin. M K & Luck. I> J L. J ccii bio! 72 (1977) 67. 19. Coyne, B & Rosenbaam, J L. J cell biol 47 (1970) 777. Received October 5. I979 Accepted November 16, 1979

Exp

Cell

RP\

127 119XO~