T-Cadherin Deficiency Is Associated with Increased Blood Pressure after Physical Activity.

T-cadherin is a regulator of blood vessel remodeling and angiogenesis, involved in adiponectin-mediated protective effects in the cardiovascular system and in skeletal muscles. GWAS study has previously demonstrated a SNP in the Cdh13 gene to be associated with hypertension. However, the role of T-cadherin in regulating blood pressure has not been experimentally elucidated. Herein, we generated Cdh13∆Exon3 mice lacking exon 3 in the Cdh13 gene and described their phenotype. Cdh13∆Exon3 mice exhibited normal gross morphology, life expectancy, and breeding capacity. Meanwhile, their body weight was considerably lower than of WT mice. When running on a treadmill, the time spent running and the distance covered by Cdh13∆Exon3 mice was similar to that of WT. The resting blood pressure in Cdh13∆Exon3 mice was slightly higher than in WT, however, upon intensive physical training their systolic blood pressure was significantly elevated. While adiponectin content in the myocardium of Cdh13∆Exon3 and WT mice was within the same range, adiponectin plasma level was 4.37-fold higher in Cdh13∆Exon3 mice. Moreover, intensive physical training augmented the AMPK phosphorylation in the skeletal muscles and myocardium of Cdh13∆Exon3 mice as compared to WT. Our data highlight a critically important role of T-cadherin in regulation of blood pressure and stamina in mice, and may shed light on the pathogenesis of hypertension.

Divergent Contribution of the Golgi Apparatus to Microtubule Organization in Related Cell Lines.

Membrane trafficking in interphase animal cells is accomplished mostly along the microtubules. Microtubules are often organized radially by the microtubule-organizing center to coordinate intracellular transport. Along with the centrosome, the Golgi often serves as a microtubule-organizing center, capable of nucleating and retaining microtubules. Recent studies revealed the role of a special subset of Golgi-derived microtubules, which facilitates vesicular traffic from this central transport hub of the cell. However, proteins essential for microtubule organization onto the Golgi might be differentially expressed in different cell lines, while many potential participants remain undiscovered. In the current work, we analyzed the involvement of the Golgi complex in microtubule organization in related cell lines. We studied two cell lines, both originating from green monkey renal epithelium, and found that they relied either on the centrosome or on the Golgi as a main microtubule-organizing center. We demonstrated that the difference in their Golgi microtubule-organizing activity was not associated with the well-studied proteins, such as CAMSAP3, CLASP2, GCC185, and GMAP210, but revealed several potential candidates involved in this process.

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.

Positioning of endoplasmic reticulum exit sites around the Golgi depends on BicaudalD2 and Rab6 activity.

The endoplasmic reticulum (ER) is involved in biogenesis, modification and transport of secreted and membrane proteins. The ER membranes are spread throughout the cell cytoplasm as well as the export domains known as ER exit sites (ERES). A subpopulation of ERES is centrally localized proximal to the Golgi apparatus. The significance of this subpopulation on ER-to-Golgi transport remains unclear. Transport carriers (TCs) form at the ERES via a COPII-dependent mechanism and move to Golgi on microtubule (MT) tracks. It was shown previously that ERES are distributed along MTs and undergo chaotic short-range movements and sporadic rapid long-range movements. The long-range movements of ERES are impaired by either depolymerization of MTs or inhibition of dynein, suggesting that ERES central concentration is mediated by dynein activity. We demonstrate that the processive movements of ERES are frequently coupled with the TC departure. Using the Sar1a[H79G]-induced ERES clustering at the perinuclear region, we identified BicaudalD2 (BicD2) and Rab6 as components of the dynein adaptor complex which drives perinuclear ERES concentration at the cell center. BicD2 partially colocalized with ERES and with TC. Peri-Golgi ERES localization was significantly affected by inhibition of BicD2 function with its N-terminal fragment or inhibition of Rab6 function with its dominant-negative mutant. Golgi accumulation of secretory protein was delayed by inhibition of Rab6 and BicD2. Thus, we conclude that a BicD2/Rab6 dynein adaptor is required for maintenance of Golgi-associated ERES. We propose that Golgi-associated ERES may enhance the efficiency of the ER-to-Golgi transport.

On the interaction of ribosomal protein RPL22e with microtubules.

Microtubule (MT) protein preparations often contain components of the translation machinery, including ribosome proteins. To understand the biological meaning of it we studied the interaction of ribosomal protein RPL22e with the MT. We found that bacteria expressed purified RPL22e-GFP-6His did co-sediment with brain tubulin MTs with 1.3 µM dissociation coefficient. Such a KD is comparable to some specific MT-associated proteins. Distinct in vitro interaction of RPL22e-GFP with MTs was also observed by TIRF microscopy. In real-time assay, RPL22e-GFP molecules stayed bound to MTs for several seconds, and 15% of them demonstrated random-walk along MTs with diffusion coefficient 0.03 µ2 /s. Deletion of basic areas of RPL22e did not have an impact on KD , and deletion of acidic tail slightly increased association with MTs. Interestingly, the deletion of acidic tail increased diffusion coefficient as well. The interaction of RPL22e with MTs is hardly noticeable in vivo in cultured cells, probably since a significant part of the protein is incorporated into the ribosomes. The mobility of ribosomal protein on the MTs probably prevents its interfering with MT-dependent transport and could ameliorate its transport to the nucleus.

The effect of Intravenous Immunoglobulin (IVIG) on \textit{ex vivo} activation of human leukocytes.

INTRODUCTION: Intravenous immunoglobulin (IVIG) has been widely used to treat various conditions, including inflammatory and autoimmune diseases. IVIG has been shown to have a direct influence on neutrophils, eosinophils and lymphocytes. However, many aspects IVIG's effect on neutrophils activation still remain unknown. OBJECTIVE: To evaluate the effect of IVIG on PMA-induced activation of neutrophils, with and without priming with TNF-α, in a series of in vitro experiments performed on whole blood. RESULTS: Our data coincided with well-known literature indicating that the effect of phorbol 12-myristate 13-acetate (PMA) on human leukocytes includes activation of neutrophils, monocytes and eosinophils, increase of chemiluminescence (CL) and induction of netosis, resulting in assembly of traps. In presence of IVIG (10 mg/mL), CL was reduced by 25% in response to PMA compared to PMA-induced leukocyte activation without IVIG. Decreasing IVIG concentration to 1 mg/mL and below did not inhibit PMA-induced activation of CL.PMA-induced activation after TNF-α priming resulted in approximately 50% increase of amplitude of CL response to PMA. Moreover, maximum CL was reached by minute 5, which was more rapid than in the absence of TNF-α-priming (in this case maximum CL was reached by minute 15).The IVIG concentrations did not affect morphological changes of leukocytes after sequential addition of TNF-α and PMA. IVIG had no effect on leukocyte content and on PMA-induced CL of primed leukocytes.Addition of IVIG under TNF-α priming significantly increased the number of traps in the smears in response to PMA activation. Of note, such an increase in the number of traps was depended on the IVIG concentration in plasma. CONCLUSION: In conclusion, we suggest that IVIG is able to reduce the degradation of traps under priming with TNF-α. Moreover, IVIG might switch the activation of primed leukocytes to netosis.

Comparative analysis of sphingomyelin synthase 1 gene expression at the transcriptional and translational levels in human tissues.

Sphingomyelin synthase 1 (SMS1) catalyses the biosynthesis of sphingomyelin in eukaryotic cells. We have previously determined the structure of the SGMS1 gene encoding this enzyme and a number of its alternative transcripts. Here, we describe a study of the expression of the full-length SMS1 protein and the sum of the alternative transcripts encoding this protein in human tissues. The SMS1 protein and mRNA levels in tissues differed significantly and were not correlated, implying the active post-transcriptional regulation of SMS1 protein expression. The putative truncated isoforms of the SMS1 protein, which are encoded by a number of alternative transcripts, were not detected by immunoblotting and thus may be absent or present in only small amounts.

CK1 activates minus-end-directed transport of membrane organelles along microtubules.

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.