The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate–dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21^Cip1 accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.     

Untangling a Decline in Tropical Forest Resilience: Constraints on the Sustainability of Shifting Cultivation Across the Globe

Shifting cultivators depend on forest biomass inputs to nourish their crops. For them, forest resilience has an immediate impact: it affects crop productivity. A decline in the rate of recovery following shifting cultivation would ultimately affect local, regional and global carbon budgets, with feedbacks to climate. Yet the long-term impacts of shifting cultivation have been quantified in only six locations. In this study, we reanalyze data from these locations to determine whether the rate of biomass recovery is the same from cycle to cycle. Further, using case studies in Southern Yucatan, Mexico and West Kalimantan, Indonesia, we investigate the ecological and socioeconomic factors that affect forest resilience and thus determine whether or not shifting cultivation is sustainable. The reanalysis links aboveground biomass recovery following shifting cultivation to site productivity, forest age, fallow length, history of cultivation, and soil texture. Across locations, biomass accumulation rate declines by 9.3 percent with each cycle of shifting cultivation. Per cycle change in biomass accumulation rate is significantly more negative in younger forests and forests that experience a shorter fallow period. However, more detailed analyses for two case studies suggest that a purely ecological framework is of limited effectiveness in explaining variability in the effect of repeated shifting cultivation. Rather, socioeconomic factors such as migration, subsidies, roads, and settlement history can alter the outcome of shifting cultivation by limiting the accumulation and use of local knowledge.     

Liprin-α1 affects the distribution of low-affinity β1 integrins and stabilizes their permanence at the cell surface

Integrins mediate the interaction between cells and extracellular matrix by assembling adhesive structures that need to be dynamically modulated to allow cell motility. We have recently identified liprin-α1 as an essential regulator of integrin dynamics required for efficient cell motility. Here we investigated the effects of liprin-α1 expression on β1 integrin receptors. We found that increased levels of liprin-α1 affected the localization of inactive, low-affinity integrins, while increasing the average size of β1 integrin-positive focal adhesions. Although a direct interaction between β1 integrins and liprin-α1 could not be revealed biochemically, a striking colocalization between redistributed inactive β1 integrins and liprin-α1 was observed. The tight association of overexpressed and endogenous liprin-α1 to the cytoplasmic side of the ventral plasma membrane suggested a possible role of liprin in stabilizing integrin receptors at the cell surface. In support of this hypothesis, we demonstrated an inhibitory effect of liprin overexpression on antibody-induced β1 integrin internalization. On the other hand, depletion of endogenous liprin-α by small interfering RNA increased the rate of integrin internalization. Overall, these results support the hypothesis that liprin-α1 exerts its action on focal adhesion turnover by influencing the localization and stability of integrin receptors at the cell surface.     

Cellular analysis of host cell infection by different developmental stages of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular parasite that infects phagocytic and non-phagocytic mammalian cells by a complex process that appears to involve several discrete steps. Even though the infection process was described many years ago, the molecular mechanisms involved remain poorly understood. As fluorescent proteins have proven to be excellent tools for live-cell imaging, we used EGFP- and DsRed1-1-transfected trypomastigotes, amastigotes and epimastigotes to study the infection process in living cells. Contrary to what has been reported, our results showed that epimastigotes are as infective as trypomastigotes and amastigotes. Besides, differences in replication, differentiation and parasite release times were observed among the stages. Our results suggest that the different developmental stages use distinct attachment and invasion mechanisms. We propose that fluorescent-based plasmid expression systems are good models for studying the infection process of intracellular microorganisms and could offers insights about the molecular mechanisms involved.     

Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.     

Acquisition of Temozolomide Chemoresistance in Gliomas Leads to Remodeling of Mitochondrial Electron Transport Chain

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.