Identification of a unique endoplasmic retention motif in the Xenopus GIRK5 channel and its contribution to oocyte maturation.

G protein-activated inward-rectifying potassium (K+ ) channels (Kir3/GIRK) participate in cell excitability. The GIRK5 channel is present in Xenopus laevis oocytes. In an attempt to investigate the physiological role of GIRK5, we identified a noncanonical di-arginine endoplasmic reticulum (ER) retention motif (KRXY). This retention motif is located at the N-terminal region of GIRK5, coded by two small exons found only in X. laevis and X. tropicalis. These novel exons are expressed through use of an alternative transcription start site. Mutations in the sequence KRXY produced functional channels and induced progesterone-independent oocyte meiotic progression. The chimeric proteins enhanced green fluorescent protein (EGFP)-GIRK5-WT and the EGFP-GIRK5K13AR14A double mutant, were localized to the ER and the plasma membrane of the vegetal pole of the oocyte, respectively. Silencing of GIRK5 or blocking of this channel by external barium prevented progesterone-induced meiotic progression. The endogenous level of GIRK5 protein decreased through oocyte stages in prophase I augmenting by progesterone. In conclusion, we have identified a unique mechanism by which the expression pattern of a K+ channel evolved to control Xenopus oocyte maturation.

Method for the Direct Fabrication of Polyacrylamide Hydrogels with Controlled Stiffness in Polystyrene Multiwell Plates for Mechanobiology Assays.

Polyacrylamide (PAA) hydrogels are now widely used in mechanobiology because the well-defined available protocols allow a robust and reproducible control of substrate stiffness within a physiological range. However, several assays require hydrogels inside traditional plastic substrates and the current methods remain relatively tedious. Here, we present a simple and direct fabrication technique that successfully attaches PAA hydrogels inside polystyrene multiwell plates and Petri dishes of different sizes. It permits a control of the Young's modulus of the gels, within the desired range for mechanobiology. Some critical steps, that had to be overcome to guarantee protein conjugation and cell attachment, are detailed, as they differ from the standardized preparation on glass substrates. To validate our process, we demonstrated that HepG2 and 3T3L1 cell lines as well as primary hepatocytes seeded on PAA gels of different stiffnesses in plastics showed a mechanical response identical to the cells cultured on traditional gels.

Building a microfluidic cell culture platform with stiffness control using Loctite 3525 glue.

The study of mechanotransduction signals and cell response to mechanical properties requires designing culture substrates that possess some, or ideally all, of the following characteristics: (1) biological compatibility and adhesive properties, (2) stiffness control or tunability in a dynamic mode, (3) patternability on the microscale and (4) integrability in microfluidic chips. The most common materials used to address cell mechanotransduction are hydrogels, due to their softness. However, they may be impractical when complex scaffolds are sought and they lack viscous dissipative properties that are very important in mechanobiology. In this work, we show that an off-the-shelf, biocompatible photosensitive glue, Loctite 3525, may be used readily in mechanobiology assays without any special treatment prior to fabrication of cell culture platforms. Despite a high (MPa) stiffness easily tunable by UV exposure time at a fixed dose, 3T3 fibroblasts showed a response to the mechanics of the material similar to that obtained on much softer (kPa) hydrogels. Loctite's viscous dissipation properties indeed seemed to be responsible for such cell mechanical response, as suggested by recent works where more complex two-phase hydrogels were employed. More interestingly, it was possible to stiffen soft Loctite substrates by post-exposing them during cell culture, to observe changes in cell spreading caused by a dynamic stiffness modification. Thanks to Loctite 3525's patternability, micropillars were also fabricated to demonstrate the compatibility with traction force microscopy studies. Finally, the glue was used as an excellent adhesion layer for hydrogels on glass or PDMS, without the need for additional treatment, enabling the easy fabrication of microfluidic chips integrating hydrogels.

Progress on the Use of Commercial Digital Optical Disc Units for Low-Power Laser Micromachining in Biomedical Applications.

The development of organ-on-chip and biological scaffolds is currently requiring simpler methods for microstructure biocompatible materials in three dimensions, to fabricate structural and functional elements in biomaterials, or modify the physicochemical properties of desired substrates. Aiming at addressing this need, a low-power CD-DVD-Blu-ray laser pickup head was mounted on a programmable three-axis micro-displacement system in order to modify the surface of polymeric materials in a local fashion. Thanks to a specially-designed method using a strongly absorbing additive coating the materials of interest, it has been possible to establish and precisely control processes useful in microtechnology for biomedical applications. The system was upgraded with Blu-ray laser for additive manufacturing and ablation on a single platform. In this work, we present the application of these fabrication techniques to the development of biomimetic cellular culture platforms thanks to the simple integration of several features typically achieved with traditional, less cost-effective microtechnology methods in one step or through replica-molding. Our straightforward approach indeed enables great control of local laser microablation or polymerization for true on-demand biomimetic micropatterned designs in transparent polymers and hydrogels and is allowing integration of microfluidics, microelectronics, surface microstructuring, and transfer of superficial protein micropatterns on a variety of biocompatible materials.

Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron.

Several potassium (K(+)) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K(+) channels allow sodium reabsorption in the proximal tubule (PT), K(+) recycling and K(+) reabsorption in the thick ascending limb (TAL) and K(+) secretion and K(+) reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K(+) to function as a secretory K(+) channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K(+) channels. Our results expand the repertoire of K(+) channels that contribute to K(+) homeostasis to include the Kv1 family.

The polarization of the G-protein activated potassium channel GIRK5 to the vegetal pole of Xenopus laevis oocytes is driven by a di-leucine motif.

The G protein-coupled inwardly-rectifying potassium channels (known as GIRK or Kir3) form functional heterotetramers gated by G-ßγ subunits. GIRK channels participate in heart rate modulation and neuronal postsynaptic inhibition in mammals. In Xenopus laevis oocytes, GIRK5 is a functional homomultimer. Previously, we found that phosphorylation of a tyrosine (Y16) at its N-terminus downregulates the surface expression of GIRK5. In this work, we elucidated the subcellular localization and trafficking of GIRK5 in oocytes. Several EGFP-GIRK5 chimeras were produced and an ECFP construct was used to identify the endoplasmic reticulum (ER). Whereas GIRK5-WT was retained in the ER at the animal pole, the phospho-null GIRK5-Y16A was localized to the vegetal pole. Interestingly, a construct with an N-terminal Δ25 deletion produced an even distribution of the channel in the whole oocyte. Through an alanine-scan, we identified an acidic cluster/di-leucine sorting-signal recognition motif between E17 and I22. We quantified the effect of each amino acid residue within this di-leucine motif in determining the distribution of GIRK5 to the animal and vegetal poles. We found that Y16 and I22 contributed to functional expression and were dominant in the polarization of GIRK5. We thus conclude that the N-terminal acidic di-leucine motif of GIRK5 determines its retention and polarized trafficking within Xl oocytes.