A Single Conserved Residue Mediates Binding of the Ribonucleotide Reductase Catalytic Subunit RRM1 to RRM2 and Is Essential for Mouse Development

The ribonucleotide reductase (RNR) complex, composed of a catalytic subunit (RRM1) and a regulatory subunit (RRM2), is thought to be a rate-limiting enzymatic complex for the production of nucleotides. In humans, the Rrm1 gene lies at 11p15.5, a tumor suppressor region, and RRM1 expression in cancer has been shown to predict responses to chemotherapy. Nevertheless, whether RRM1 is essential in mammalian cells and what the effects of its haploinsufficiency are remain unknown. To model RNR function in mice we used a mutation previously described in Saccharomyces cerevisiae (Rnr1-W688G) which, despite being viable, leads to increased interaction of the RNR complex with its allosteric inhibitor Sml1. In contrast to yeast, homozygous mutant mice carrying the Rrm1 mutation (Rrm1^WG/WG) are not viable, even at the earliest embryonic stages. Proteomic analyses failed to identify proteins that specifically bind to the mutant RRM1 but revealed that, in mammals, the mutation prevents RRM1 binding to RRM2. Despite the impact of the mutation, Rrm1^WG/+ mice and cells presented no obvious phenotype, suggesting that the RRM1 protein exists in excess. Our work reveals that binding of RRM1 to RRM2 is essential for mammalian cells and provides the first loss-of-function model of the RNR complex for genetic studies.     

A Highlights from MBoC Selection: Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell''s height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.     

CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.     

Identification of Campylobacter pylori in gastric biopsy smears

The use of gastric biopsy imprint smears to diagnose Campylobacter pylori was compared with the use of tissue sections and cultures. Multiple gastric biopsies were taken from the mucosa of 42 patients during endoscopy. Imprint smears were prepared from the samples used to make tissue sections; other samples were used for microbiologic culture. There was a good concordance (93%) between the morphologic diagnosis of C pylori in the air-dried, Giemsa-stained smears and the tissue sections; the cytologic preparations were clearly positive in six cases (14%) whose sections contained low numbers of the organisms. There was a concordance of 83% between the combined morphologic techniques and the bacteriologic culture. Six positive cases were detected only by the morphologic techniques while one positive case was detected only by bacteriologic culture. C pylori was identified in one or more preparations of the antral biopsy specimens in 23 (55%) of the 42 cases, including 23 (74%) of the 31 cases with a final diagnosis of gastritis or ulcer. These results show the usefulness of the cytologic study of gastric biopsy smears in diagnosing C pylori infections.     

Control of water-use efficiency by florigen

A major issue in modern agriculture is water loss through stomata during photosynthetic carbon assimilation. In water-limited ecosystems, annual plants have strategies to synchronize their growth and reproduction to the availability of water. Some species or ecotypes of flowers are early to ensure that their life cycles are completed before the onset of late season terminal drought (“drought escape”). This accelerated flowering correlates with low water-use efficiency (WUE). The molecular players and physiological mechanisms involved in this coordination are not fully understood. We analyzed WUE using gravimetry, gas exchange, and carbon isotope discrimination in florigen deficient (sft mutant), wild-type (Micro-Tom), and florigen over-expressing (SFT-ox) tomato lines. Increased florigen expression led to accelerated flowering time and reduced WUE. The low WUE of SFT-ox was driven by higher stomatal conductance and thinner leaf blades. This florigen-driven effect on WUE appears be independent of abscisic acid (ABA). Our results open a new avenue to increase WUE in crops in an ABA-independent manner. Manipulation of florigen levels could allow us to produce crops with a life cycle synchronized to water availability.