Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.
Peripheral nerve injury results in persistent motor deficits, even after the nerve regenerates and muscles are reinnervated. This lack of functional recovery is partly explained by brain and spinal cord circuit alterations triggered by the injury, but the mechanisms are generally unknown. One example of this plasticity is the die-back in the spinal cord ventral horn of the projections of proprioceptive axons mediating the stretch reflex (Ia afferents). Consequently, Ia information about muscle length and dynamics is lost from ventral spinal circuits, degrading motor performance after nerve regeneration. Simultaneously, there is activation of microglia around the central projections of peripherally injured Ia afferents, suggesting a possible causal relationship between neuroinflammation and Ia axon removal. Therefore, we used mice (both sexes) that allow visualization of microglia (CX3CR1-GFP) and infiltrating peripheral myeloid cells (CCR2-RFP) and related changes in these cells to Ia synaptic losses (identified by VGLUT1 content) on retrogradely labeled motoneurons. Microgliosis around axotomized motoneurons starts and peaks within 2 weeks after nerve transection. Thereafter, this region becomes infiltrated by CCR2 cells, and VGLUT1 synapses are lost in parallel. Immunohistochemistry, flow cytometry, and genetic lineage tracing showed that infiltrating CCR2 cells include T cells, dendritic cells, and monocytes, the latter differentiating into tissue macrophages. VGLUT1 synapses were rescued after attenuating the ventral microglial reaction by removal of colony stimulating factor 1 from motoneurons or in CCR2 global KOs. Thus, both activation of ventral microglia and a CCR2-dependent mechanism are necessary for removal of VGLUT1 synapses and alterations in Ia-circuit function following nerve injuries.
The role of Rab coupling protein (RCP) has not been previously investigated in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore RCP protein expression and its clinicopathological significance in SCCHN. RCP protein expression in 95 SCCHN samples, 18 vocal nodule epithelia and 16 leukoplakia epithelia samples was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Our data indicated that vocal nodule epithelia, leukoplakia epithelia and SCCHN showed a gradual increase in the expression of RCP protein. RCP overexpression was significantly associated with T classification, clinical staging, lymph node metastasis and recurrence. Survival analysis revealed that a high RCP expression was significantly correlated with shorter overall survival and disease-free survival. In conclusion, RCP protein may contribute to the malignant progression of SCCHN, and serves as a novel prognostic marker in patients with SCCHN.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
This review is based in part on a roundtable discussion session: "Physiological roles for heterotypic/heteromeric channels" at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.
5-hydroxymethylcytosine (5hmC) is enriched in brain and has been recognized as an important DNA modification. However, the roles of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in stress-induced response have yet to be elucidated. Here, we show that chronic restraint stress (CRS) induced depression-like behavior in mice and resulted in a 5hmC reduction in prefrontal cortex (PFC). We found that loss of Tet1 (Tet1 KO) led to resistance to CRS, whereas loss of Tet2 (Tet2 KO) increased the susceptibility of mice to CRS. Genome-wide 5hmC profiling identified the phenotype-associated stress-induced dynamically hydroxymethylated loci (PA-SI-DhMLs), which are strongly enriched with hypoxia-induced factor (HIF) binding motifs. We demonstrated the physical interaction between TET1 and HIF1α induced by CRS and revealed that the increased HIF1α binding under CRS is associated with SI-DhMLs. These results suggest that TET1 could regulate stress-induced response by interacting with HIF1α. The roles of 5-hydroxymethylcytosine (5hmC) and its writers, Tet proteins, in stress-induced response remain unclear. Cheng et al. show that Tet1 knockout mice exhibit resistance, whereas Tet2 knockout mice have increased susceptibility to stress. Biochemical and genome-wide analyses suggest that Tet1 could regulate stress-induced response by interacting with Hif1α.
by
Christoph Brockmann;
Sharon Soucek;
Sonja I. Kuhlmann;
Katherine Mills-Lujan;
Seth Kelly;
Ji-Chun Yang;
Nahid Iglesias;
Francoise Stutz;
Anita Corbett;
David Neuhaus;
Murray Stewart
Polyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit. Structural coherence is lost in the RNA-binding compromised Nab2-C437S mutant, which also suppresses the rat8-2 allele of RNA helicase Dbp5. Structure-guided Nab2 variants indicate that dbp5(rat8-2) suppression is more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to affinity for polyadenosine-RNA. These results indicate that, in addition to modulating polyA tail length, Nab2 has an unanticipated function associated with generating export-competent mRNPs, and that changes within fingers 5-7 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.
Nuclear pore complexes are composed of ∼30 different proteins, each present at the pore in multiple copies. Together these proteins create specialized channels that convey cargo between the cytoplasm and the nuclear interior. With the building blocks of nuclear pores identified, one challenge is to decipher how these proteins are coordinately produced and assembled into macromolecular pore structures with each cell division. Specific individual pore proteins and protein cofactors have been probed for their role in the assembly process, as well as certain kinases that add a layer of regulation via the phosphorylation status of nucleoporins. Other posttranslational modifications are candidates for coordinating events of pore assembly as well. In this study of two pore-associated small ubiquitin-like modifier (SUMO) proteases, sentrin/SUMO-specific protease 1 (SENP1) and SENP2, we observe that many nucleoporins are misloc alized and, in some cases, reduced in level when SENP1 and SENP2 are codepleted. The pore complexes present under these conditions are still capable of transport, although the kinetics of specific cargo is altered. These results reveal a new role for the pore-associated SENPs in nucleoporin homeostasis and in achieving proper configuration of the nuclear pore complex.
Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson’s disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trak1 that causes hypertonia in mice. Moreover, elevated Trak1 protein expression is associated with several types of cancers and variants in Trak1 are linked to childhood absence epilepsy in humans. Despite the importance of Trak1 in health and disease, the mechanisms of Trak1 action remain unclear and the pathogenic effects of Trak1 mutation are unknown. Here we report that Trak1 has a crucial function in regulation of mitochondrial fusion. Depletion of Trak1 inhibits mitochondrial fusion, resulting in mitochondrial fragmentation, whereas overexpression of Trak1 elongates and enlarges mitochondria. Our analyses revealed that Trak1 interacts and colocalizes with mitofusins on the outer mitochondrial membrane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trak1 is required for stress-induced mitochondrial hyperfusion and pro-survival response. We found that hypertonia-associated mutation impairs Trak1 mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trak1 as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochondrial dynamics to hypertonia pathogenesis.
Rare genetic diseases are the result of a continuous forward genetic screen that nature is conducting on humans. Here, we present epistemological and systems biology arguments highlighting the importance of studying these rare genetic diseases. We contend that the expanding catalog of mutations in ∼4,000 genes, which cause ∼6,500 diseases and their annotated phenotypes, offer a wide landscape for discovering fundamental mechanisms required for human development and involved in common diseases. Rare afflictions disproportionately affect the nervous system in children, but paradoxically, the majority of these disease-causing genes are evolutionarily ancient and ubiquitously expressed in human tissues. We propose that the biased prevalence of childhood rare diseases affecting nervous tissue results from the topological complexity of the protein interaction networks formed by ubiquitous and ancient proteins encoded by childhood disease genes. Finally, we illustrate these principles discussing Menkes disease, an example of the discovery power afforded by rare diseases.