SummaryIntegrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor a3b1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a ratelimiting role in cell migration and integrin trafficking. In STX6-depleted cells, a3b1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in a3b1 integrin delivery to the cell surface, and endocytosed a3b1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.

Deciphering the histone code has illustrated that acetylation or methylation on the same residue can have analogous or opposing roles. However, little is known about the interplay between these post-translational modifications (PTMs) on the same nonhistone residues. We have recently discovered that N-terminal acetyltransferases (NATs) and N-terminal methyltransferases (NRMTs) can have overlapping substrates and identified myosin regulatory light chain 9 (MYL9) as the first confirmed protein to occur in either α-amino-methylated (Nα-methyl) or α-amino-acetylated (Nα-acetyl) states in vivo. Here we aim to determine if these PTMs function similarly or create different MYL9 proteoforms with distinct roles. We use enzymatic assays to directly verify MYL9 is a substrate of both NRMT1 and NatA and generate mutants of MYL9 that are exclusive for Nα-acetylation or Nα-methylation. We then employ eukaryotic cell models to probe the regulatory functions of these Nα-PTMs on MYL9. Our results show that, contrary to prevailing dogma, neither of these modifications regulate the stability of MYL9. Rather, exclusive Nα-acetylation promotes cytoplasmic roles of MYL9, while exclusive Nα-methylation promotes the nuclear role of MYL9 as a transcription factor. The increased cytoplasmic activity of Nα-acetylated MYL9 corresponds with increased phosphorylation at serine 19, a key MYL9 activating PTM. Increased nuclear activity of Nα-methylated MYL9 corresponds with increased DNA binding. Nα-methylation also results in a decrease of interactions between the N-terminus of MYL9 and a host of cytoskeletal proteins. These results confirm that Nα-acetylation and Nα-methylation differentially affect MYL9 function by creating distinct proteoforms with different internal PTM patterns and binding properties.

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Endometrial dysfunction affects approximately 1% of infertile women, and there is currently no standard therapy for improving fertility treatment outcomes in these patients. In our study, we utilized a rodent model of thin endometrium to test whether intrauterine application of adipose-derived stromal vascular fraction cells (SVF) could improve morphological and physiological markers of endometrial receptivity. Using anhydrous ethanol, endometrial area and gland density were significantly reduced in our model of thin endometrium. Application of SVF was associated with a 29% reduction in endometrial vascular endothelial growth factor (VEGF) expression and significant increases in uterine artery systolic/diastolic velocity ratios and resistance index values, suggesting reduced diastolic microvascular tone. However, no significant improvements in endometrial area or gland density were observed following SVF treatment. 3D confocal imaging demonstrated poor engraftment of SVF cells into recipient tissue, which likely contributed to the negative results of this study. We suspect modified treatment protocols utilizing adjuvant estrogen and/or tail vein cell delivery may improve SVF retention and therapeutic response in subsequent studies. SVF is an easily-obtainable cell product with regenerative capability that may have a future role in the treatment of infertile women with endometrial dysfunction.

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