Precursor types predict the stability of neuronal branches

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. While branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the altered stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase related protein 3 (PLPPR3/PRG2) or neurons treated with Netrin-1. Rather than altering branch stability directly, PLPPR3 and Netrin-1 boost a ‘filopodia branch program’ on axons, thereby indirectly initiating more long-lived branches. In summary, we propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models as exemplified in this study.

Cellular Localization of Exogenous Cry1Ab/c and its Interaction with Plasma Membrane Ca2+-ATPase in Transgenic Rice

The cellular localization of exogenous proteins expressed in transgenic crops not only determines their stability, but also their effects on crop growth and development, including under stressful conditions; however, the underlying molecular mechanisms remain unknown. Here, we determined the cellular distribution of exogenously expressed Cry1Ab/c protein in insect-resistant transgenic rice Huahui-1 (HH1) cells through subcellular localization, immunohistochemistry, immunofluorescence, and western blot analyses. Interaction between the Cry1Ab/c protein and the preliminarily screened endogenous plasma membrane protein Ca2+-ATPase was investigated through yeast two-hybrid, bimolecular fluorescence complementation (BIFC), and co-immunoprecipitation analyses. The potential interaction mechanism was analyzed by comparing the cellular localization and interaction sites between Cry1Ab/c and Ca2+-ATPase. Phenotypic indices and Ca2+-ATPase activity, which may be regulated by the Cry1Ab/c–Ca2+-ATPase interaction, were determined in transgenic HH1 and the parental line Minghui-63 under stress-free and salt-stress conditions. The results showed that Cry1Ab/c was not only distributed in the cytoplasm and nucleus but was also distributed on the plasma membrane, where it interacted with plasma membrane Ca2+-ATPase. This interaction partially retain plasma membrane protein Ca2+-ATPase in the nucleus by a BIFC experiment and thus may affect Ca2+-ATPase activity on the membrane by altering the cellular location of the protein. Consistently, our results confirmed that the presence of Cry1Ab/c in the transgenic HH1 resulted in a reduction in Ca2+-ATPase activity as well as causing detrimental effects on plant phenotype, including significantly reduced plant height and biomass, compared to parental MH63; and that these detrimental effects were more pronounced under salt stress conditions, impacting the salt resistance of the transgenic plants. We suggest that the Cry1Ab/c–Ca2+-ATPase interaction may explain the plasma membrane localization of Cry1Ab/c, which lacks a signal peptide and a transmembrane domain, and the adverse effects of Cry1Ab/c expression on the growth and development of transgenic HH1 plants under salt stress. This information may clarify the molecular mechanisms of these unintended effects and demonstrate the feasibility of evaluating the success and performance of genetic modification of commercially vital crops.

Ankylosis homologue mediates cellular efflux of ATP, not pyrophosphate

The plasma membrane protein Ankylosis Homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints, but we recently showed that ANKH releases large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. This ATP is converted extracellularly into PPi and AMP by the ectoenzyme Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANK. We now addressed this question by determining the effect of a complete absence of ENPP1 on ANKH-dependent extracellular PPi concentrations. Introduction of ANKH in ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi seen in ENPP1-proficient cells.Ank-activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained < 2.5% of the PPi found in bones of wild type mice, showing that Enpp1-activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60-70 % of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification, therefore do so by extruding NTPs rather than PPi itself.

Comparative phylogenetic analysis of CBL reveals the gene family evolution and functional divergence in Saccharum spontaneum

Background The identification and functional analysis of genes that improve tolerance to low potassium stress in S. spontaneum is crucial for breeding sugarcane cultivars with efficient potassium utilization. Calcineurin B-like (CBL) protein is a calcium sensor that interacts with specific CBL-interacting protein kinases (CIPKs) upon plants’ exposure to various abiotic stresses. Results In this study, nine CBL genes were identified from S. spontaneum. Phylogenetic analysis of 113 CBLs from 13 representative plants showed gene expansion and strong purifying selection in the CBL family. Analysis of CBL expression patterns revealed that SsCBL01 was the most commonly expressed gene in various tissues at different developmental stages. Expression analysis of SsCBLs under low K+ stress indicated that potassium deficiency moderately altered the transcription of SsCBLs. Subcellular localization showed that SsCBL01 is a plasma membrane protein and heterologous expression in yeast suggested that, while SsCBL01 alone could not absorb K+, it positively regulated K+ absorption mediated by the potassium transporter SsHAK1. Conclusions This study provided insights into the evolution of the CBL gene family and preliminarily demonstrated that the plasma membrane protein SsCBL01 was involved in the response to low K+ stress in S. spontaneum.

Overexpression of MsRCI2A, MsRCI2B, and MsRCI2C in Alfalfa (Medicago sativa L.) Provides Different Extents of Enhanced Alkali and Salt Tolerance Due to Functional Specialization of MsRCI2s

Rare cold-inducible 2/plasma membrane protein 3 (RCI2/PMP3) genes are ubiquitous in plants and belong to a multigene family whose members respond to a variety of abiotic stresses by regulating ion homeostasis and stabilizing membranes, thus preventing damage. In this study, the expression of MsRCI2A, MsRCI2B, and MsRCI2C under high-salinity, alkali and ABA treatments was analyzed. The results showed that the expression of MsRCI2A, MsRCI2B, and MsRCI2C in alfalfa (Medicago sativa L.) was induced by salt, alkali and ABA treatments, but there were differences between MsRCI2 gene expression under different treatments. We investigated the functional differences in the MsRCI2A, MsRCI2B, and MsRCI2C proteins in alfalfa (Medicago sativa L.) by generating transgenic alfalfa plants that ectopically expressed these MsRCI2s under the control of the CaMV35S promoter. The MsRCI2A/B/C-overexpressing plants exhibited different degrees of improved phenotypes under high-salinity stress (200 mmol.L–1 NaCl) and weak alkali stress (100 mmol.L–1 NaHCO3, pH 8.5). Salinity stress had a more significant impact on alfalfa than alkali stress. Overexpression of MsRCI2s in alfalfa caused the same physiological response to salt stress. However, in response to alkali stress, the three proteins encoded by MsRCI2s exhibited functional differences, which were determined not only by their different expression regulation but also by the differences in their regulatory relationship with MsRCI2s or H+-ATPase.

Precursor types predict the stability of neuronal branches

Branches are critical for neuron function, generating the morphological complexity required for functional networks. They emerge from different, well-described, cytoskeletal precursor structures that elongate to branches. While branches are thought to be maintained by shared cytoskeletal regulators, our data from mouse hippocampal neurons indicate that the precursor structures trigger alternative branch maintenance mechanisms with differing stabilities. While branches originating from lamellipodia or growth cone splitting events collapse soon after formation, branches emerging from filopodia persist. Furthermore, compared to other developing neurites, axons stabilise all branches and preferentially initiate branches from filopodia. These differences explain the decreased stability of branches we observe in neurons lacking the plasma membrane protein phospholipid phosphatase related protein 3 (PLPPR3/PRG2). Rather than altering branch stability directly, PLPPR3 boosts a ‘filopodia branch program’ on axons, thereby indirectly initiating more long-lived branches. We propose that studies on branching should distinguish overall stabilising effects from effects on precursor types, ideally using multifactorial statistical models as exemplified in this study.

Pectin Dependent Cell Adhesion Restored by a Mutant Microtubule Organizing Membrane Protein

The cellulose- and pectin-rich plant cell wall defines cell structure, mediates defense against pathogens, and facilitates plant cell adhesion. An adhesion mutant screen of Arabidopsis hypocotyls identified a new allele of QUASIMODO2 (QUA2), a gene required for pectin accumulation and whose mutants have reduced pectin content and adhesion defects. A suppressor of qua2 was also isolated and describes a null allele of SABRE (SAB), which encodes a previously described plasma membrane protein required for longitudinal cellular expansion that organizes the tubulin cytoskeleton. sab mutants have increased pectin content, increased levels of expression of pectin methylesterases and extensins, and reduced cell surface area relative to qua2 and Wild Type, contributing to a restoration of cell adhesion.

Plasma Membrane Protein Nce102 Modulates Morphology and Function of the Yeast Vacuole

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.