A genetic program boosts mitochondrial function to power macrophage tissue invasion

SUMMARYMetabolic adaptation to changing demands underlies homeostasis. During inflammation or metastasis, cells leading migration into challenging environments require an energy boost, however what controls this capacity is unknown. We identify a previously unstudied nuclear protein, Atossa, as changing metabolism in Drosophila melanogaster immune cells to promote tissue invasion. Atossa’s vertebrate orthologs, FAM214A-B, can fully substitute for Atossa, indicating functional conservation from flies to mammals. Atossa increases mRNA levels of Porthos, an unstudied RNA helicase and two metabolic enzymes, LKR/SDH and GR/HPR. Porthos increases translation of a gene subset, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Respiration measurements and metabolomics indicate that Atossa and Porthos powers up mitochondrial oxidative phosphorylation to produce sufficient energy for leading macrophages to forge a path into tissues. As increasing oxidative phosphorylation enables many crucial physiological responses, this unique genetic program may modulate a wide range of cellular behaviors beyond migration.

ZNF423 orthologs are highly constrained in vertebrates but show domain-level plasticity across invertebrate lineages

ZNF423 encodes 30 C2H2 zinc fingers that bind DNA and a variety of lineage- and signal-dependent transcription factors. ZNF423 genetic variants are proposed to cause neurodevelopmental and ciliopathy-related disorders in humans. Mouse models show midline brain defects, including cerebellar vermis hypoplasia, and defects in adipogenesis. Here I show strong protein sequence constraint among 165 vertebrate orthologs. In contrast, orthologs from invertebrate lineages, spanning larger time intervals, show substantial differences in zinc finger number, arrangement, and identity. A terminal zinc finger cluster common among other lineages was independently lost in vertebrates and insects. Surprisingly, a moderately-constrained non-C2H2 sequence with potential to form a C4-class zinc finger is a previously-unrecognized conserved feature of nearly all identified homologs. These results highlight evolutionary dynamics of a likely signal integration node across species with distinct developmental strategies and body plans. Functions of the newly identified C4-like sequence and lineage-specific fingers remain to be studied.

Epidermal control of axonal attachment via β-spectrin and the GTPase-activating protein TBC-10 prevents axonal degeneration

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/β-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/β-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that β-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/β-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.

PCR amplification and sequence analysis of the rat Sox3 gene

The Sox3 gene is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Despite the completion of a rat genome sequencing project, only a partial sequence of the rat Sox3 gene has been available in the public database. Using PCR, sequencing, and bioinformatics tools, in this study we have determined the complete coding sequence of the rat Sox3 gene encoding 449 amino acids. Comparative analysis of rat and human SOX3 proteins revealed a high degree of conservation. Identification of the rat Sox3 gene sequence would help in understanding the biological roles of this gene and provide insight into evolutionary relationships with vertebrate orthologs.

Zebrafish Meis functions to stabilize Pbx proteins and regulate hindbrain patterning

Homeodomain-containing Hox proteins regulate segmental identity in Drosophila in concert with two partners known as Extradenticle (Exd) and Homothorax (Hth). These partners are themselves DNA-binding, homeodomain proteins, and probably function by revealing the intrinsic specificity of Hox proteins. Vertebrate orthologs of Exd and Hth, known as Pbx and Meis (named for a myeloid ecotropic leukemia virus integration site), respectively, are encoded by multigene families and are present in multimeric complexes together with vertebrate Hox proteins. Previous results have demonstrated that the zygotically encoded Pbx4/Lazarus (Lzr) protein is required for segmentation of the zebrafish hindbrain and proper expression and function of Hox genes. We demonstrate that Meis functions in the same pathway as Pbx in zebrafish hindbrain development, as expression of a dominant-negative mutant Meis results in phenotypes that are remarkably similar to that of lzr mutants. Surprisingly, expression of Meis protein partially rescues the lzr– phenotype. Lzr protein levels are increased in embryos overexpressing Meis and are reduced for lzr mutants that cannot bind to Meis. This implies a mechanism whereby Meis rescues lzr mutants by stabilizing maternally encoded Lzr. Our results define two functions of Meis during zebrafish hindbrain segmentation: that of a DNA-binding partner of Pbx proteins, and that of a post-transcriptional regulator of Pbx protein levels.

Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

The Evolution of the Neural Basic Helix-Loop-Helix Proteins

Basic Helix-Loop-Helix (bHLH) transcription factors control various aspects of the formation of the nervous system in the metazoans. In Drosophila some bHLH (such as the achaete-scuteatonal, and amos genes) act as proneural genes, directing ectodermal cells toward a neural fate. Their vertebrate orthologs, however, probably do not assume such a neural determination function, but rather control the decision made by neural precursors to generate neurons and not glial cells, as well as the progression of neuronal precursors toward differentiation into mature neurons. The proneural function of Drosophila bHLH genes may be an innovation that occurs in the evolutive lineage that leads to arthropods. In addition, although neural bHLH appear to be involved in the specification of neuronal identities, they probably do not confer by themselves neuronal type-specific properties to the cells. Rather, neural bHLH allow neural cells to correctly interpret specification and positional cues provided by other factors. Although bHLH genes are often expressed in complementary subsets of neural cells and/or expressed sequentially in those cells, the coding regions of the various neural bHLH appear largely interchangeable. We propose that the specific expression patterns have been acquired, following gene duplications, by subfunctional-ization, i.e., the partitioning of ancestral expression patterns among the duplicates and, by extension, we propose that subfunctionalization is a key process to understand the evolution of neural bHLH genes.

FAST-2 Is a Mammalian Winged-Helix Protein Which Mediates Transforming Growth Factor β Signals

ABSTRACT The mechanisms by which transforming growth factor β (TGF-β) and related ligands regulate transcription remain poorly understood. The winged-helix (WH) transcription factor fork head activin signal transducer 1 (FAST-1) was identified as a mediator of activin signaling in Xenopus embryos (X. Chen, M. J. Rubock, and M. Whitman, Nature 383:691–696, 1996). We have cloned a novel WH gene from the mouse which shares many properties with FAST-1. We find that this gene, which we call FAST-2, is able to mediate transcriptional activation by TGF-β. FAST-2 also interacts directly with Smad2, a cytoplasmic protein which is translocated to the nucleus in response to TGF-β, and forms a multimeric complex with Smad2 and Smad4 on the activin response element, a high-affinity binding site for FAST-1. Analysis of the sequences of FAST-1 and FAST-2 reveals substantial protein sequence divergence compared to known vertebrate orthologs in the WH family. This suggests that FAST-2 represents a new WH gene related to FAST-1, which functions to mediate TGF-β signals in mammals. We have also examined the structure of the FAST-2 gene and find that it overlaps with a kinesin motor protein gene. The genes are transcribed in opposite orientations, and their transcripts overlap in the 3′ untranslated region.