Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching.

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)--fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.

Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation.

A series of gusA transcriptional fusion vectors is described for Bacillus subtilis (Bs). The series includes a vector for use with the amyE system of Shimotsu and Henner [Gene 43 (1986) 85-94], an integrative vector and vectors that provide gusA or gusA neo cassettes. The gusA fusions are compatible with lacZ fusion vectors that are widely used with Bs, and gusA and lacZ fusions are expressed at similar levels. beta-Glucuronidase (beta Glu) and beta-galactosidase (beta Gal) do not exhibit any cross-reactivity, there is very little endogenous beta Glu activity in Bs, and there is no indication of mutation to high-level expression. We have use strains containing both gusA and lacZ fusions to compare the times of expression of different genes during sporulation.

Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis.

Sporulation of Bacillus subtilis requires the coordinated expression of two separate developmental programs in the mother cell and forespore compartments by sigma E and sigma F, respectively. This coordination is maintained through the action of cross-regulatory factors that control the activities of the various sporulation-specific sigma factors. We present here the isolation and characterization of one such cross-regulatory factor, the spoIIR gene. Using a genetic screen, we have isolated four mutant alleles of spoIIR. These mutants were isolated as expressing sigma F-directed genes but not sigma E-directed genes. The block in sigma E-directed gene expression in spoIIR mutants was caused by an inability to process pro-sigma E to its active form. Cloning and characterization of the spoIIR gene determined that its transcription is directed by sigma F. Thus, SpoIIR is required for linking the activation of sigma E to the activation of sigma F and coordinating the initiation of the two developmental programs required to form a spore.

Analysis of the role of prespore gene expression in the compartmentalization of mother cell-specific gene expression during sporulation of Bacillus subtilis.

A hallmark of sporulation of Bacillus subtilis is the formation of two distinct cells by an asymmetric division. The development programs in these two cells involve the compartmentalized activities of sigma E in the larger mother cell and of sigma F in the smaller prespore. Activation of sigma E requires expression of the sigma F-directed gene spoIIR. By immunofluorescence microscopy of a strain containing a spoIIR-lacZ fusion, we have shown that spoIIR is transcribed exclusively in the prespore. By placing spoIIR under the control of PspoIIE, it was possible to express spoIIR before the spore septum was formed. Strains containing the PspoIIE-spoIIR construct activated sigma E only in the mother cell in organisms that underwent the asymmetric sporulation division. Thus, compartmentalization of sigma E activity did not require the compartmentalization of spoIIR expression. Nor did the compartmentalization of sigma E require SpoIIAA, SpoIIAB, sigma F, or sigma F-dependent transcription, all of which are required for prespore-specific gene expression. It is inferred that although sigma F and sigma E direct compartmentalized gene expression, neither of these sigma factors, nor the genes under their control, directs the process of compartmentalization.

Suppression of TGA mutations in the Bacillus subtilis spoIIR gene by prfB mutations.

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.

Enhancing leptin response by preventing SH2-containing phosphatase 2 interaction with Ob receptor.

Leptin is an adipocyte-derived cytokine that regulates food intake and body weight via interaction with its Ob receptor (ObR). Serum leptin levels are chronically elevated in obese humans, suggesting that obesity may be associated with leptin resistance and the inability to generate an adequate ObR response. Evidence suggests that transcriptional activation of target genes by STAT3 (signal transducer and activator of transcription) in the hypothalamus is a critical pathway that mediates leptin's action. Herein we report that activation of ObR induces the tyrosine phosphorylation of the tyrosine phosphatase SH2-containing phosphatase 2 (SHP-2) and demonstrate that Tyr986 within the ObR cytoplasmic domain is essential to mediate phosphorylation of SHP-2 and binding of SHP-2 to ObR. Surprisingly, mutation of Tyr986 to Phe, which abrogates SHP-2 phosphorylation and binding to the receptor, dramatically increases gene induction mediated by STAT3. Our findings indicate that SHP-2 is a negative regulator of STAT3-mediated gene induction after activation of ObR and raise the possibility that blocking the interaction of SHP-2 with ObR could overcome leptin resistance by boosting leptin's weight-reducing effects in obese individuals.