Chl4p and iml3p are two new members of the budding yeast outer kinetochore

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.     

Kinetochore protein interactions and their regulation by the Aurora kinase Ipl1p

Although there has been a recent explosion in the identification of budding yeast kinetochore components, the physical interactions that underlie kinetochore function remain obscure. To better understand how kinetochores attach to microtubules and how this attachment is regulated, we sought to characterize the interactions among kinetochore proteins, especially with respect to the microtubule-binding Dam1 complex. The Dam1 complex plays a crucial role in the chromosome-spindle attachment and is a key target for phospho-regulation of this attachment by the Aurora kinase Ipl1p. To identify protein-protein interactions involving the Dam1 complex, and the effects of Dam1p phosphorylation state on these physical interactions, we conducted both a genome-wide two-hybrid screen and a series of biochemical binding assays for Dam1p. A two-hybrid screen of a library of 6000 yeast open reading frames identified nine kinetochore proteins as Dam1p-interacting partners. From 113 in vitro binding reactions involving all nine subunits of the Dam1 complex and 32 kinetochore proteins, we found at least nine interactions within the Dam1 complex and 19 potential partners for the Dam1 complex. Strikingly, we found that the Dam1p-Ndc80p and Dam1p-Spc34p interactions were weakened by mutations mimicking phosphorylation at Ipl1p sites, allowing us to formulate a model for the effects of phosphoregulation on kinetochore function.     

Characterization of peptide-amphiphiles possessing cellular activation sequences

Numerous approaches have been described for modifying biomaterials to incorporate extracellular matrix components. "Peptide-amphiphiles", whereby monoalkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to (a) form specific molecular architecture with enhanced stability and (b) promote cell adhesion, spreading, and signaling. The present study has examined the use of chimeric peptide-amphiphiles for inducing protein-like structures and peptide-amphiphile mixtures for enhancing surface bioactivity. The alpha-helical propensity of a 21 residue peptide, incorporating the SPARC(119-122) angiogenesis-inducing sequence and either unmodified or acylated with a C(6), C(10), C(14), C(16), C(18), C(18:1), or C(18:1-OH) monoalkyl hydrocarbon chain, has been examined. Peptide and peptide-amphiphile structures were characterized by circular dichroism and one- and two-dimensional NMR spectroscopic techniques. The 21 residue peptide alone does not form a distinct structure in solution, whereas N-terminal acylation by monoalkyl hydrocarbon chains results in the 21 residue peptide-amphiphile adopting a predominantly alpha-helical structure in solution. The thermal stability of the alpha-helix increases with increasing hydrocarbon chain length. The SPARC(119-122) peptide-amphiphiles were then screened for promotion of endothelial cell adhesion and spreading. The greatest activity was achieved by using a mixture of the alpha-helical SPARC(119-122) peptide-amphiphile, a triple-helical peptide-amphiphile incorporating the alpha2beta1 integrin binding site from type I collagen, and a pseudolipid. The pseudolipid is most likely required for a spatial distribution of the peptide-amphiphiles that allows for optimal cellular interactions. Overall, we have found that incorporation of bioactive sequences within peptide-amphiphiles results in the induction of an ordered structure of the bioactive sequence and that mixtures of peptide-amphiphiles can be used to promote endothelial cell behaviors comparable to extracellular matrix components.     

Fibrobacter succinogenes S85 ferments ball-milled cellulose as fast as cellobiose until cellulose surface area is limiting

Fibrobacter succinogenes S85 grew rapidly on cellobiose (0.31 h⁻¹) and the absolute rate of increase in fermentation acids was 0.68 h⁻¹. Cultures that were provided with ball-milled cellulose initially produced fermentation acids and microbial protein as fast as those provided with cellobiose, but the absolute cellulose digestion rate eventually declined. If the inoculum size was increased, the kinetics decayed from first to zero order (with respect to cells) even sooner, but in each case the absolute rate declined after only 20 to 30% of the cellulose had been fermented. Congo red binding indicated that the cellulose surface area of individual cellulose particles was not decreasing, and the transition of ball-milled cellulose digestion corresponded with the appearance of unbound cells in the culture supernatant. When bound cells from partially digested cellulose were removed and the cellulose was re-incubated with a fresh inoculum, the initial absolute fermentation rate was as high as the one observed for undigested cellulose and cellobiose. Based on these results, cellulose digestion by F. succinogenes S85 appears to be constrained by cellulose surface area rather than cellulase activity per se. Received: 19 January 2000 / Received revision: 18 April 2000 / Accepted: 1 May 2000     

Identification of the (183)RWTNNFREY(191) region as a critical segment of matrix metalloproteinase 1 for the expression of collagenolytic activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.     

Degradation of the upper pulvinus in modern and fossil leaves of Cercis (Fabaceae)

Identification of fossil leaf impressions as Cercis has been questioned based upon the presence or absence of a pulvinus at the base of the lamina (upper pulvinus). In the present study, leaves of Cercis canadensis were examined before and after abscission to explore the degradation processes that could occur prior to fossilization, and the North American record for fossil foliage of Cercis was revised accordingly. Results for C. canadensis indicate that: (1) the pulvinus consists largely of tissues with nonlignified cells (a wide cortex, a nonlignified fiber sheath, phloem, and pith) that degrade rapidly after leaf abscission, (2) the lignified xylem tissue that remains in the pulvinus after degradation is in brittle strands, (3) the pulvinus degrades at a faster rate than the lamina or the petiole, and (4) the degraded pulvinus cushion leaves a semicircular pattern on the lamina. From examination of fossils as well as extant species, we: (1) demonstrated that in fossils, the upper pulvinus can show a greater degree of degradation than the adjoining petiole or lamina tissue, suggesting the degradation of upper pulvinus tissue is similar in modern vs. fossil specimens, (2) defined numerous other laminar characters that can be used in conjunction with, or in the absence of, an upper pulvinus to confirm the presence of Cercis in the fossil record, and (3) showed from those criteria that the earliest known North American fossil leaf record for Cercis, from a specimen newly reported in the present study, is from the middle Miocene Succor Creek flora of Oregon.