SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims: Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.<p></p> Methods: Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.<p></p> Key Results: VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.<p></p> Conclusions: These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis

Using monoclonal antibodies to label living root hairs: a novel tool for studying cell wall microarchitecture and dynamics in <i>Arabidopsis</i>

Background&lt;p&gt;&lt;/p&gt; The Arabidopsis root hair represents a valuable cell model for elucidating polar expansion mechanisms in plant cells and the overall biology of roots. The deposition and development of the cell wall is central to the root hair expansion apparatus. During this process, incorporation of specific wall polymers into the growing wall architecture constitutes a critical spatio-temporal event that controls hair size and growth rate and one that is closely coordinated with the cell’s endomembrane, cytoskeletal and signal transduction apparatuses.&lt;p&gt;&lt;/p&gt; Results&lt;p&gt;&lt;/p&gt; In this study, the protocol for live cell labeling of roots with monoclonal antibodies that bind to specific wall polymers is presented. This method allows for rapid assessment of root hair cell wall composition during development and assists in describing changes to cell wall composition in transgenic mutant lines. Enzymatic “unmasking” of specific polymers prior to labeling allows for refined interpretation of cell wall chemistry. Live cell immunofluorescence data may also be correlated with transmission electron microscopy-based immunogold labeling.&lt;p&gt;&lt;/p&gt; Conclusions&lt;p&gt;&lt;/p&gt; Live Arabidopsis root hairs may be labeled with cell wall polymer-specific antibodies. This methodology allows for direct visualization of cell wall dynamics throughout development in stable transgenic plant lines. It also provides an important new tool in the elucidation of the specific interactions occurring between membrane trafficking networks, cytoskeleton and the cell wall deposition/remodeling mechanism

Clathrin Heavy Chain subunits coordinate endo- and exocytic traffic and affect stomatal movement

The current model for vesicular traffic to and from the plasma membrane is accepted but the molecular requirements for this coordination are not well defined. We have identified the has1 mutant, which has a stomatal function defect, as a clathrin heavy chain 1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane is sensitive to CHC1 and CHC2 function in seedling roots, and that chc mutants have physiological defects in stomatal function and plant growth that have not been previously described. These findings suggest that clathrin supports specific functions of multiple cell types. Stomata movement and gas exchange is altered in chc mutants, indicating clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which are also similar to those of the secretory SNARE mutant, syp121. The syp121 and chc mutants have impaired endo- and exocytosis compared to wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endo- and exocytosis, and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis

Vocal Responses to Perturbations in Voice Auditory Feedback in Individuals with Parkinson's Disease

One of the most common symptoms of speech deficits in individuals with Parkinson's disease (PD) is significantly reduced vocal loudness and pitch range. The present study investigated whether abnormal vocalizations in individuals with PD are related to sensory processing of voice auditory feedback. Perturbations in loudness or pitch of voice auditory feedback are known to elicit short latency, compensatory responses in voice amplitude or fundamental frequency.Twelve individuals with Parkinson's disease and 13 age- and sex-matched healthy control subjects sustained a vowel sound (/α/) and received unexpected, brief (200 ms) perturbations in voice loudness (±3 or 6 dB) or pitch (±100 cents) auditory feedback. Results showed that, while all subjects produced compensatory responses in their voice amplitude or fundamental frequency, individuals with PD exhibited larger response magnitudes than the control subjects. Furthermore, for loudness-shifted feedback, upward stimuli resulted in shorter response latencies than downward stimuli in the control subjects but not in individuals with PD.The larger response magnitudes in individuals with PD compared with the control subjects suggest that processing of voice auditory feedback is abnormal in PD. Although the precise mechanisms of the voice feedback processing are unknown, results of this study suggest that abnormal voice control in individuals with PD may be related to dysfunctional mechanisms of error detection or correction in sensory feedback processing

Diversity of retromer-mediated vesicular trafficking pathways in plants

The plant endomembrane system is organized and regulated by large gene families that encode proteins responsible for the spatiotemporal delivery and retrieval of cargo throughout the cell and to and from the plasma membrane. Many of these regulatory molecules form functional complexes like the SNAREs, exocyst, and retromer, which are required for the delivery, recycling, and degradation pathways of cellular components. The functions of these complexes are well conserved in eukaryotes, but the extreme expansion of the protein subunit families in plants suggests that plant cells require more regulatory specialization when compared with other eukaryotes. The retromer is associated with retrograde sorting and trafficking of protein cargo back towards the TGN and vacuole in plants, while in animals, there is new evidence that the VPS26C ortholog is associated with recycling or ‘retrieving’ proteins back to the PM from the endosomes. The human VPS26C was shown to rescue vps26c mutant phenotypes in Arabidopsis thaliana, suggesting that the retriever function could be conserved in plants. This switch from retromer to retriever function may be associated with core complexes that include the VPS26C subunit in plants, similar to what has been suggested in other eukaryotic systems. We review what is known about retromer function in light of recent findings on functional diversity and specialization of the retromer complex in plants

Synergy Among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth

Vesicle exocytosis underpins signaling and development in plants and is vital for wall remodeling during cell expansion. Vesicle tethering and fusion are thought to occur sequentially, tethering mediated by the exocyst and fusion driven by assembly of SNARE protein complexes. Interactions between these two protein complexes are known, although insights into their functional consequences are largely unexplored. We now identify a clear hierarchy of interactions leading to secretion in Arabidopsis. Mating-based split-ubiquitin screens and in vivo FRET analyses showed that exocyst EXO70 subunits bind preferentially with cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distributions and suppressed vesicle traffic like the dominant-negative SYP121deltaC, consistent with the epistasis of exo70A1 over the exo70A1syp121 double mutant. However, the exo70A1vamp721 mutant showed a strong synergistic suppression of growth. These data are best explained by a spatiotemporal hierarchy of exocyst recruitment with the R-SNARE to the plasma membrane, plausibly with the VAMP721 longin domain as a nexus for binding

Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition

Directional cell movement is universally required for tissue morphogenesis. Although it is known that cell/matrix interactions are essential for directional movement in heart development, the mechanisms governing these interactions require elucidation. Here we demonstrate that a novel protein/protein interaction between blood vessel epicardial substance (Bves) and N-myc downstream regulated gene 4 (NDRG4) is critical for regulation of epicardial cell directional movement, as disruption of this interaction randomizes migratory patterns. Our studies show that Bves/NDRG4 interaction is required for trafficking of internalized fibronectin through the “autocrine extracellular matrix (ECM) deposition” fibronectin recycling pathway. Of importance, we demonstrate that Bves/NDRG4-mediated fibronectin recycling is indeed essential for epicardial cell directional movement, thus linking these two cell processes. Finally, total internal reflectance fluorescence microscopy shows that Bves/NDRG4 interaction is required for fusion of recycling endosomes with the basal cell surface, providing a molecular mechanism of motility substrate delivery that regulates cell directional movement. This is the first evidence of a molecular function for Bves and NDRG4 proteins within broader subcellular trafficking paradigms. These data identify novel regulators of a critical vesicle-docking step required for autocrine ECM deposition and explain how Bves facilitates cell-microenvironment interactions in the regulation of epicardial cell–directed movement

WISE/NEOWISE observations of Active Bodies in the Main Belt

We report results based on mid-infrared photometry of 5 active main belt objects (AMBOs) detected by the Wide-field Infrared Survey Explorer (WISE) spacecraft. Four of these bodies, P/2010 R2 (La Sagra), 133P/Elst-Pizarro, (596) Scheila, and 176P/LINEAR, showed no signs of activity at the time of the observations, allowing the WISE detections to place firm constraints on their diameters and albedos. Geometric albedos were in the range of a few percent, and on the order of other measured comet nuclei. P/2010 A2 was observed on April 2-3, 2010, three months after its peak activity. Photometry of the coma at 12 and 22 {\mu}m combined with ground-based visible-wavelength measurements provides constraints on the dust particle mass distribution (PMD), dlogn/dlogm, yielding power-law slope values of {\alpha} = -0.5 +/- 0.1. This PMD is considerably more shallow than that found for other comets, in particular inbound particle fluence during the Stardust encounter of comet 81P/Wild 2. It is similar to the PMD seen for 9P/Tempel 1 in the immediate aftermath of the Deep Impact experiment. Upper limits for CO2 & CO production are also provided for each AMBO and compared with revised production numbers for WISE observations of 103P/Hartley 2.Comment: 32 Pages, including 5 Figure

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). Methods: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. Results: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Conclusions: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affectcartilage development

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the roleof Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre,Osx1-Cre, and Col2a1-Cre drivers, respectively. Wild-type and conditional knockout mice were phenotypically assessed by grossmorphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated usingRNA-sequencing, histologic evaluation, and western blotting. Aged mice with Ezh2 deficiency were also evaluated for prematuredevelopment of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age, although caused no other gross developmentaleffects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3lysine 27 (H3K27me3) and altered differentiation in vitro. RNA-seq analysis revealed enrichment of an osteogenic gene expressionprofile in Ezh2 deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes withoutinducing excessive hypertrophy or premature osteoarthritis in vivo. In summary, loss of Ezh2 reduced H3K27me3 levels, increased expression of osteogenic genes in chondrocytes, and resulted ina transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondralossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage-commitment