Long-lived mitochondrial cristae proteins in mouse heart and brain

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)–based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.     

Mapping bone cell distributions to assess ontogenetic origin of primate midfacial form

Midfacial reduction in primates has been explained as a byproduct of other growth patterns, especially the convergent orbits. This is at once an evolutionary and developmental explanation for relatively short snouts in most modern primates. Here, we use histological sections of perinatal nonhuman primates (tamarin, tarsier, loris) to investigate how orbital morphology emerges during ontogeny in selected primates compared to another euarchontan (Tupaia glis). We annotated serial histological sections for location of osteoclasts or osteoblasts, and used these to create three‐dimensional “modeling maps” showing perinatal growth patterns of the facial skeleton. In addition, in one specimen we transferred annotations from histological sections to CT slices, to create a rotatable 3D volume that shows orbital modeling. Our findings suggest that growth in the competing orbital and neurocranial functional matrices differs among species, influencing modeling patterns. Distinctions among species are observed in the frontal bone, at a shared interface between the endocranial fossa and the orbit. The medial orbital wall is extensively resorptive in primates, whereas the medial orbit is generally depositional in Tupaia. As hypothesized, the orbital soft tissues encroach on available interorbital space. However, eye size cannot, by itself, explain the extent of reduction of the olfactory recess. In Loris, the posterior portion of medial orbit differed from the other primates. It showed evidence of outward drift where the olfactory bulb increased in cross‐sectional area. We suggest the olfactory bulbs are significant to orbit position in strepsirrhines, influencing an expanded interorbital breadth at early stages of development. Am J Phys Anthropol 154:424–435, 2014. © 2014 Wiley Periodicals, Inc.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Traction force microscopy in rapidly moving cells reveals separate roles for ROCK and MLCK in the mechanics of retraction

Retraction is a major rate-limiting step in cell motility, particularly in slow moving cell types that form large stable adhesions. Myosin II dependent contractile forces are thought to facilitate detachment by physically pulling up the rear edge. However, retraction can occur in the absence of myosin II activity in cell types that form small labile adhesions. To investigate the role of contractile force generation in retraction, we performed traction force microscopy during the movement of fish epithelial keratocytes. By correlating changes in local traction stress at the rear with the area retracted, we identified four distinct modes of retraction. “Recoil” retractions are preceded by a rise in local traction stress, while rear edge is temporarily stuck, followed by a sharp drop in traction stress upon detachment. This retraction type was most common in cells generating high average traction stress. In “pull” type retractions local traction stress and area retracted increase concomitantly. This was the predominant type of retraction in keratocytes and was observed mostly in cells generating low average traction stress. “Continuous” type retractions occur without any detectable change in traction stress, and are seen in cells generating low average traction stress. In contrast, to many other cell types, “release” type retractions occur in keratocytes following a decrease in local traction stress. Our identification of distinct modes of retraction suggests that contractile forces may play different roles in detachment that are related to rear adhesion strength. To determine how the regulation of contractility via MLCK or Rho kinase contributes to the mechanics of detachment, inhibitors were used to block or augment these pathways. Modulation of MLCK activity led to the most rapid change in local traction stress suggesting its importance in regulating attachment strength. Surprisingly, Rho kinase was not required for detachment, but was essential for localizing retraction to the rear. We suggest that in keratocytes MLCK and Rho kinase play distinct, complementary roles in the respective temporal and spatial control of rear detachment that is essential for maintaining rapid motility.     

Active coping toward predatory stress is associated with lower corticosterone and progesterone plasma levels and decreased methylation in the medial amygdala vasopressin system

An active coping style displayed under stress – which involves proactive investigatory responses toward environmental threats – has been associated with reduced vulnerability to psychiatric illness. However, the neurobiological determinants of coping styles are not well understood. When rats are exposed to a naturalistic stressor (cat fur) in a group, some individuals in the group show robust active investigation of the stimulus while others show a passive response involving retreat, immobility and close aggregation with conspecifics. Here we explored endocrine and epigenetic correlates of these contrasting coping styles. Male Wistar rats (n = 48) were exposed to cat fur in groups of 4 and the passive and active responders were identified and assessed for endocrine and epigenetic differences. Three days after the final cat fur exposure, active responders had substantially lower plasma levels of corticosterone and progesterone than passive responders. Plasma and testicular testosterone levels did not differ between active and passive responders. Active responders had markedly less methylation of the AVP CGCG promoter region located at base 4970 in the posterodorsal region of the medial amygdala but did not differ in the methylation status of the CCGG sequence located at base 2243. This is in agreement with prior research suggesting that AVP and progesterone act in opposition within the medial amygdala to modulate stress-related behaviors. The present study reports striking endocrine and epigenetic differences between active and passive responders, providing insight into potential systems involved in the manifestation of differing coping styles.     

Differences in Survival and Growth Among Tropical Rain Forest Pioneer Tree Seedlings in Relation to Canopy Openness and Herbivory

Although differences in canopy openness, herbivory and their interaction may promote species coexistence, how these factors affect pioneer tree species and potentially limit growth, and survival has been poorly studied, particularly in tropical South Asia. We monitored the effect of canopy openness and herbivore damage on seedling survival and growth of 960 individuals of six pioneer tree species: Dillenia triquetra, Macaranga indica, Macaranga peltata, Schumacheria castaneifolia, Trema orientalis, and Wendlandia bicuspidata. Seedlings were placed in four gap‐understory positions—center, outer gap edge, inner forest edge, and understory—in four large, natural gaps within the Sinharaja World Heritage Reserve, Sri Lanka. Canopy openness positively affected survival probability beyond the 550‐d experiment, while herbivory decreased survival and was highest in understory conditions. The relative order of species survival stayed fairly consistent between gap‐understory positions and followed their known shade tolerance rankings. When averaged across all experimental conditions, T. orientalis had the lowest survival probability estimate beyond the 550‐d experiment (0.05), but the greatest capacity for growth where it successfully established, while the species with highest averaged survival probability (0.79), D. triquetra, showed the lowest growth. One species, W. bicuspidata, responded positively to herbivory by re‐sprouting. Coexistence of D. triquetra, T. orientalis, and W. bicuspidata can be explained by a trade‐off among species in survival, growth, and response to herbivory. In addition to variation in canopy light environment, herbivory may be important in determining pioneer species distribution through fine‐scale niche partitioning and should be carefully considered in reforestation efforts.     

Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

More than 50 Helicobacter pylori genes are predicted to encode outer membrane proteins (OMPs), but there has been relatively little experimental investigation of the H. pylori cell surface proteome. In this study, we used selective biotinylation to label proteins localized to the surface of H. pylori, along with differential detergent extraction procedures to isolate proteins localized to the outer membrane. Proteins that met multiple criteria for surface-exposed outer membrane localization included known adhesins, as well as Cag proteins required for activity of the cag type IV secretion system, putative lipoproteins, and other proteins not previously recognized as cell surface components. We identified sites of nontryptic cleavage consistent with signal sequence cleavage, as well as C-terminal motifs that may be important for protein localization. A subset of surface-exposed proteins were highly susceptible to proteolysis when intact bacteria were treated with proteinase K. Most Hop and Hom OMPs were susceptible to proteolysis, whereas Hor and Hof proteins were relatively resistant. Most of the protease-susceptible OMPs contain a large protease-susceptible extracellular domain exported beyond the outer membrane and a protease-resistant domain at the C terminus with a predicted β-barrel structure. These features suggest that, similar to the secretion of the VacA passenger domain, the N-terminal domains of protease-susceptible OMPs are exported through an autotransporter pathway. Collectively, these results provide new insights into the repertoire of surface-exposed H. pylori proteins that may mediate bacterium-host interactions, as well as the cell surface topology of these proteins.     

Reduced and reversed temperature dependence of blood oxygenation in an ectothermic scombrid fish: implications for the evolution of regional heterothermy?

Tunas (family Scombridae) are exceptional among most teleost fishes in that they possess vascular heat exchangers which allow heat retention in specific regions of the body (termed ‘regional heterothermy’). Seemingly exclusive to heterothermic fishes is a markedly reduced temperature dependence of blood–oxygen (blood–O₂) binding, or even a reversed temperature dependence where increasing temperature increases blood–O₂ affinity. These unusual binding properties have been documented in whole blood and in haemoglobin (Hb) solutions, and they are hypothesised to prevent oxygen loss from arteries to veins within the vascular heat exchangers and/or to prevent excessive oxygen unloading to the warm tissues and ensure an adequate supply of oxygen to tissues positioned efferent to the heat exchangers. The temperature sensitivity of blood–O₂ binding has not been characterised in an ectothermic scombrid (mackerels and bonitos), but the existence of the unusual binding properties in these fishes would have clear implications for their proposed association with regional heterothermy. Accordingly, the present study examined oxygenation of whole blood of the chub mackerel (Scomber japonicus) at 10, 20 and 30°C and at 0.5, 1 and 2% CO₂. Oxygen affinity was generally highest at 20°C for all levels of CO₂. Temperature-independent binding was observed at low (0.5%) CO₂, where the PO₂ at 50% blood–O₂ saturation (P ₅₀) was not statistically different at 10 and 30°C (2.58 vs. 2.78 kPa, respectively) with an apparent heat of oxygenation (∆H°) close to zero (−6 kJ mol⁻¹). The most significant temperature-mediated difference occurred at high (2%) CO₂, where the P ₅₀ at 10°C was twofold higher than that at 20°C with a corresponding ∆H° of +43 kJ mol⁻¹. These results provide clear evidence of independent and reversed open-system temperature effects on blood oxygenation in S. japonicus, and it is therefore speculated that these unusual blood–O₂ binding characteristics may have preceded the evolution of vascular heat exchangers and regional heterothermy in fishes.     

Cyclin-Dependent Kinase 1-Mediated Bcl-xL/Bcl-2 Phosphorylation Acts as a Functional Link Coupling Mitotic Arrest and Apoptosis

Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-x_L and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-x_L and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-x_L mutant but not by a phosphomimetic Bcl-x_L mutant, confirming Bcl-x_L as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.The cell division cycle is controlled by checkpoints, which ensure the fidelity of chromosome replication and segregation, as well as orderly progression through the cell cycle. If these critical events cannot be completed as scheduled, damaged cells, which might otherwise pose a threat to the organism as precancerous cells, are eliminated (16). The mitotic checkpoint, for example, produces a “prevent anaphase” signal until all the chromosomes are properly attached to kinetochores (22). Microtubule inhibitors (MTIs) and other antimitotic agents prolong the activation of this checkpoint, causing mitotic arrest, which culminates in cell death generally via intrinsic apoptosis, providing a rationale for the use of these agents as antitumor agents (20, 31). Intrinsic or mitochondrial apoptosis is regulated by the Bcl-2 family of proteins, which exhibit either pro- or antiapoptotic properties (17, 37). The BH3-only proapoptotic members act as essential initiators of intrinsic apoptosis, whereas the multidomain proapoptotic members, Bax and Bak, act as essential mediators of mitochondrial membrane permeability. Antiapoptotic Bcl-2 family members, including Bcl-x_L, Bcl-2, and Mcl-1, oppose apoptosis by binding to the proapoptotic members and neutralizing their activity.The molecular mechanisms leading to cell death in response to spindle checkpoint activation have yet to be established. Indeed, how the spindle checkpoint couples to pathways regulating cell survival and death still represents an unresolved issue in cell biology (26, 35). Nonetheless, it seems reasonable to hypothesize that signals generated in response to prolonged mitotic arrest are eventually transduced to the apoptotic machinery. In this regard, it is striking that MTIs consistently induce the phosphorylation of two key antiapoptotic proteins, Bcl-2 and Bcl-x_L, whereas other apoptotic stimuli fail to do so (9, 13, 25). The results of studies with phosphodefective mutants of Bcl-2 and Bcl-x_L indicate that phosphorylation antagonizes their antiapoptotic function (2, 33, 36), but the precise mechanism(s) has yet to be fully clarified.The identity of the kinase responsible for the extensive phosphorylation of Bcl-x_L and Bcl-2 that occurs in response to sustained spindle checkpoint activation is unresolved. Identification of this kinase is considered to be of critical importance, since it will provide insight into the molecular links between mitotic arrest and cell death, as well as the molecular mechanism of action of antimitotic drugs. Several candidates have been proposed, including Raf-1 (3), Jun N-terminal protein kinase (JNK) (2, 11, 36), protein kinase A (PKA) (32), cyclin-dependent kinase 1 (CDK1) (24), and mammalian target of rapamycin (mTOR) (4). In general, however, conclusions have been correlative or have been based on the use of kinase inhibitors tested under conditions that precluded mitotic arrest and thus indirectly blocked the effects of MTIs. Thus, strong experimental evidence supporting identification is lacking.Here we present evidence that the CDK1/cyclin B kinase complex is responsible for mitotic arrest-induced Bcl-x_L/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. The findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-x_L/Bcl-2 phosphorylation, resulting in inactivation of the antiapoptotic function of Bcl-x_L/Bcl-2. Thus, CDK1-mediated phosphorylation of antiapoptotic Bcl-2 proteins acts as a key link coupling mitotic arrest to apoptosis.