Dimerization of Hsp90 is required for in vivo function. Design and analysis of monomers and dimers

Heat shock protein 90 (Hsp90) plays a central role in signal transduction and has emerged as a promising target for anti-cancer therapeutics, but its molecular mechanism is poorly understood. At physiological concentration, Hsp90 predominantly forms dimers, but the function of full-length monomers in cells is not clear. Hsp90 contains three domains: the N-terminal and middle domains contribute directly to ATP binding and hydrolysis and the C domain mediates dimerization. To study the function of Hsp90 monomers, we used a single-chain strategy that duplicated the C-terminal dimerization domain. This novel monomerization strategy had the dual effect of stabilizing the C domain to denaturation and hindering intermolecular association of the ATPase domain. The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. The monomeric construct was also defective at ATP hydrolysis and the activation of a kinase and steroid receptor substrate in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric.     

Pisodonophis boro (ophichthidae: anguilliformes): Specialization for head‐first and tail‐first burrowing?

The rice paddy eel, Pisodonophis boro (P. boro), is of special interest because of its peculiar burrowing habits. P. boro penetrates the substrate tail-first, a technique common for ophichthids, but it is able to burrow head-first as well. P. boro exhibits three feeding modes: inertial feeding, grasping, and spinning. Rotational feeding is a highly specialized feeding mode, adopted by several elongate, aquatic vertebrates and it is likely that some morphological modifications are related to this feeding mode. The detailed morphology of the head and tail of P. boro is examined with the goal to apportion the anatomical specializations among head-first burrowing, tail-first burrowing, and rotational feeding. The reduced eyes, covered with thick corneas may be beneficial for protection during head-first burrowing, but at the same time decreased visual acuity may have an impact on other sensory systems (e.g. cephalic lateral line system). The elongated and pointed shape of the skull is beneficial for substrate penetration. The cranial bones and their joints, which are fortified, are advantageous for resisting high mechanical loads during head-first burrowing. The aponeurotic connection between epaxial and jaw muscles is considered beneficial for transferring these forces from the body to the head during rotational feeding. Hypertrophied jaw muscles facilitate a powerful bite, which is required to hold prey during spinning movements and variability in the fiber angles of subdivisions of jaw muscles may be beneficial for preventing the lower jaw from being dislodged or opened. Furthermore, firm upper (premaxillo-ethmovomerine complex) and lower jaws (with robust coronoid processes) and high neurocranial rigidity are advantageous for a solid grip to hold prey during rotational feeding. The pointed shape of the tail and the consolidated caudal skeleton are beneficial for their tail-first burrowing habits. It is quite likely that the reduction of the caudal musculature is related to the tail-first burrowing behavior because the subtle movements of the caudal fin rays are no longer required.     

MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

N-cadherin regulates cytoskeletally associated IQGAP1/ERK signaling and memory formation

Cadherin-mediated interactions are integral to synapse formation and potentiation. Here we show that N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling.     

SEL-2, the C. elegans neurobeachin/LRBA homolog, is a negative regulator of lin-12/Notch activity and affects endosomal traffic in polarized epithelial cells

The vulval precursor cells (VPCs) of Caenorhabditis elegans are polarized epithelial cells that adopt a precise pattern of fates through regulated activity of basolateral LET-23/EGF receptor and apical LIN-12/Notch. During VPC patterning, there is reciprocal modulation of endocytosis and trafficking of both LET-23 and LIN-12. We identified sel-2 as a negative regulator of lin-12/Notch activity in the VPCs, and found that SEL-2 is the homolog of two closely related human proteins, neurobeachin (also known as BCL8B) and LPS-responsive, beige-like anchor protein (LRBA). SEL-2, neurobeachin and LRBA belong to a distinct subfamily of BEACH-WD40 domain-containing proteins. Loss of sel-2 activity leads to basolateral mislocalization and increased accumulation of LIN-12 in VPCs in which LET-23 is not active, and to impaired downregulation of basolateral LET-23 in VPCs in which LIN-12 is active. Downregulation of apical LIN-12 in the VPC in which LET-23 is active is not affected. In addition, in sel-2 mutants, the polarized cells of the intestinal epithelium display an aberrant accumulation of the lipophilic dye FM4-64 when the dye is presented to the basolateral surface. Our observations indicate that SEL-2/neurobeachin/LRBA is involved in endosomal traffic and may be involved in efficient delivery of cell surface proteins to the lysosome. Our results also suggest that sel-2 activity may contribute to the appropriate steady-state level of LIN-12 or to trafficking events that affect receptor activation.     

EVA regulates thymic stromal organisation and early thymocyte development

Epithelial V-like antigen (EVA) is an immunoglobulin-like adhesion molecule identified in a screen for molecules developmentally regulated at the DN to DP progression in thymocyte development. We show that EVA is expressed during the early stages of thymus organogenesis in both fetal thymic epithelia and T cell precursors, and is progressively downregulated from day 16.5 of embryonic development. In the postnatal thymus, EVA expression is restricted to epithelial cells and is distributed throughout both cortical and medullary thymic regions. Transgenic overexpression of EVA in the thymus cortex resulted in a modified stromal environment, which elicited an increase in organ size and absolute cell number. Although peripheral T lymphocyte numbers are augmented throughout life, no imbalance either in the repertoire, or in the different T cell subsets was detected. Collectively, these data suggest a role for EVA in structural organisation of the thymus and early lymphocyte development.     

Organ-specificity and inducibility of patatin class I promoter from potato in transgenic arabidopsis plants

Patatin class I promoter (B33 promoter) is a tissue-specific potato (Solanum tuberosum L.) promoter expressing the patatin gene mainly in tubers. However, it can be induced in other organs by sucrose or light. We compared the activity of this promoter fused with the reporter gene during heterological expression in B33::GUS transgenic arabidopsis (Arabidopsis thaliana L.) plants and homological expression of the same DNA construct in potato. Promoter activity was estimated from quantification of β-glucuronidase (GUS) activity. It was shown that, during heterological expression in arabidopsis seedlings, B33 promoter manifested a tissue-specificity and inducibility, although in a different manner than during homological expression in potato. In noninduced arabidopsis seedlings, B33 promoter was most active in the roots, whereas, after induction with sucrose treatment, it became most active in cotyledons. 10 mM sucrose was sufficient for a manifold activation of B33 promoter in intact seedlings. The degree of B33 promoter induction by sucrose in arabidopsis seedlings was strictly organ-specific and increased in the following sequence: root < hypocotyl < cotyledons. 150–200 mM sucrose enhanced B33 promoter activity in cotyledons by 200 to 300 times, i.e., much stronger than in potato organs. Glucose and fructose were less efficient than sucrose. Phytohormones affecting tuber formation in potato (gibberellins, auxins, and cytokinins) did not affect significantly B33 promoter activity in arabidopsis. A lag period of approximately 6 h preceded sucrose-induced B33 promoter activation. This indicates that the patatin promoter is not the primary target for the sucrose signal. The quantitative examination of heterological expression of patatin class I promoter further clarifies its basic functional characteristics and permits a better prognosis of its behavior after transferring into other plant species.     

African trypanosomes: intracellular trafficking of host defense molecules

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.     

Morphological and Functional Divergence of the Lower Jaw Between Native and Invasive Red Foxes

Journal of Mammalian Evolution - The introduction of European red foxes in Australia in the late mid-nineteenth century has resulted in the spread of this invasive species across the continent. The...