The Myosin I SH3 Domain and TEDS Rule Phosphorylation Site are Required for In Vivo Function

The class I myosins play important roles in controlling many different types of actin-based cell movements. Dictyostelium cells either lacking or overexpressing amoeboid myosin Is have significant defects in cortical activities such as pseudopod extension, cell migration, and macropinocytosis. The existence of Dictyostelium null mutants with strong phenotypic defects permits complementation analysis as a means of exploring important functional features of the myosin I heavy chain. Mutant Dictyostelium cells lacking two myosin Is exhibit profound defects in growth, endocytosis, and rearrangement of F-actin. Expression of the full-length myoB heavy chain in these cells fully rescues the double mutant defects. However, mutant forms of the myoB heavy chain in which a serine at the consensus phosphorylation site has been altered to an alanine or in which the C-terminal SH3 domain has been removed fail to complement the null phenotype. The wild-type and mutant forms of the myoB heavy chain appeared to be properly localized when they were expressed in the myosin I null mutants. These results suggest that the amoeboid myosin I consensus phosphorylation site and SH3 domains do not play a role in the localization of myosin I, but are absolutely required for in vivo function.

The EF-hand Ca2+-binding Protein p22 Associates with Microtubules in an N-Myristoylation–dependent Manner

Proteins containing the EF-hand Ca2+-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca2+-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22–microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation–dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22’s Ca2+-binding activity, suggesting that the p22–microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Paleogene calcareous nannofossil stratigraphy of ODP Leg 120 sites

Calcareous nannofossils are abundant in the Paleogene sediments recovered during Ocean Drilling Program Leg 120. Although no continuous Paleogene section was obtained, Sites 747 through 751 complemented each other so as to provide a virtually complete composite stratigraphic section. The calcareous nannofossil biostratigraphy at Sites 747, 748, and 749 is discussed. Correlation of calcareous nannofossil biozones and magnetozones at these sites suggests some diachrony with low-latitude areas, as well as on a regional basis. Changes in calcareous nannofossil diversity throughout the Paleogene are analyzed and interpreted as reflecting major paleoclimatic events.

Calcareous nannofossil stratigraphy and datums of sediments from ODP Leg 198 sites

A relatively complete lower Paleocene to lower Oligocene sequence was recovered from the Southern High of Shatsky Rise at Sites 1209, 1210, and 1211. The sequence consists of nannofossil ooze and clay-rich nannofossil ooze. Samples from these sites have been the target of intensive calcareous nannofossil biostratigraphic investigations. Calcareous nannofossils are moderately preserved in most of the recovered sequence, which extends from nannofossil Zones CP1 to CP16. Most traditional zonal markers are present; however, the rarity and poor preservation of key species in the uppermost Paleocene and lower Eocene inhibits zonal subdivision of part of this sequence.

Calcareous nannofossil stratigraphy and key biohorizons of ODP Leg 173 sites

Six sites were drilled on the southern Iberia Abyssal Plain during Ocean Drilling Program (ODP) Leg 173. Three holes (1067A, 1068A, and 1069A) recovered Eocene sediments consisting of thinly bedded turbidite deposits with interbedded hemipelagic sediments (Bouma sequence Te) deposited near the calcite compensation depth. The hemipelagic sediments are barren of nannofossils, necessitating the use of the turbidite deposits to erect an Eocene biostratigraphy for these holes. Moderately preserved, diverse assemblages of nannofossils were recovered from silty clays (Bouma sequence Td) and poorly preserved, less diverse assemblages were recovered from sandy/silty clays (Bouma sequence Tc). Hole 1067A has a continuous record of sedimentation (Subzones CP9a-CP14a) and Holes 1068A and 1069A have similar continuous records (Subzones CP9a-CP12a), although all holes contain barren intervals. Holes 1067A, 1068A, 1069A, 900A (ODP Leg 149), and 398D (Deep Sea Drilling Project Leg 47B) display a similar increase in mass accumulation rates in the lowermost middle Eocene. A reliable Eocene biostratigraphy has been erected using nannofossil data from turbidite sequences, allowing for correlation between Iberia Abyssal Plain sites.