Deletion of MP/ARF5 domains III and IV reveals a requirement for Aux/IAA regulation in Arabidopsis leaf vascular patterning

Combinatorial interactions of AUXIN RESPONSE FACTORs (ARFs) and auxin/indole acetic acid (Aux/IAA) proteins through their common domains III and IV regulate auxin responses, but insight into the functions of individual proteins is still limited. As a new tool to explore this regulatory network, we generated a gain-of-function ARF genotype by eliminating domains III and IV from the functionally well-characterized ARF MONOPTEROS(MP)/ARF5. This truncated version of MP, termed MPΔ, conferred complementing MP activity, but also displayed a number of semi-dominant traits affecting auxin signaling and organ patterning. In MPΔ, the expression levels of many auxin-inducible genes, as well as rooting properties and vascular tissue abundance, were enhanced. Lateral organs were narrow, pointed and filled with parallel veins. This effect was epistatic over the vascular hypotrophy imposed by certain Aux/IAA mutations. Further, in MPΔ leaves, failure to turn off the procambium-selecting gene PIN1 led to the early establishment of oversized central procambial domains and very limited subsequent lateral growth of the leaf lamina. We conclude that MPΔ can selectively uncouple a single ARF from regulation by Aux/IAA proteins and can be used as a genetic tool to probe auxin pathways and explore leaf development.     

Evolution and functional cross‐talk of protein post‐translational modifications

Protein post‐translational modifications (PTMs) allow the cell to regulate protein activity and play a crucial role in the response to changes in external conditions or internal states. Advances in mass spectrometry now enable proteome wide characterization of PTMs and have revealed a broad functional role for a range of different types of modifications. Here we review advances in the study of the evolution and function of PTMs that were spurred by these technological improvements. We provide an overview of studies focusing on the origin and evolution of regulatory enzymes as well as the evolutionary dynamics of modification sites. Finally, we discuss different mechanisms of altering protein activity via post‐translational regulation and progress made in the large‐scale functional characterization of PTM function.     

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300

Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3′-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.