Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells.

MAIN CONCLUSION: PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

Pharmaceutical Analysis in the Plant Endomembrane System.

Vesicle trafficking is an essential cellular process conserved in eukaryotes to precisely transport proteins to their destinations. The plant endomembrane system plays a pivotal role in orchestrating this vesicle-mediated protein transport process, making its study essential for a comprehensive understanding of plant growth and development. Pharmaceutical analysis proves highly useful in investigating the plant endomembrane system. To facilitate further studies in this area, we present a summary of several commonly used chemical inhibitors in this chapter, providing a practical resource for researchers interested in the plant endomembrane system.

Monitoring the Intracellular Trafficking of Virus-Induced Structures and Intercellular Spread of Viral Infection in Plants Using Endomembrane Trafficking Pathway-Specific Chemical Inhibitor and Organelle-Selective Fluorescence Dye.

Infection by positive-strand RNA viruses induces extensive remodeling of the host endomembrane system in favor of viral replication and movement. The integral membrane protein 6K2 of potyviruses induces the formation of membranous virus replication vesicles at the endoplasmic reticulum exit site (ERES). The intracellular trafficking of 6K2-induced vesicles along with microfilaments requires the vesicular transport pathway, actomyosin motility system, and possibly post-Golgi compartments such as endosomes as well. Recent studies have shown that endocytosis is essential for the intracellular movement of potyviruses from the site of viral genome replication/assembly site to plasmodesmata (PD) to enter neighboring cells. In this chapter, we describe a detailed protocol of how to use endomembrane trafficking pathway-specific chemical inhibitors and organelle-selective fluorescence dye to study the trafficking of potyviral proteins and potyvirus-induced vesicles and to unravel the role of endocytosis and the endocytic pathway in potyvirus infection in Nicotiana benthamiana plants.

Kidney Function Reserve Capacity in Early and Later Stage Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: It is assumed that in autosomal dominant polycystic kidney disease (ADPKD), kidney function remains in the normal range for several decades because of hyperfiltration of remnant nephrons. In this study, we investigate the extent to which patients with ADPKD hyperfilter. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In this cross-sectional study, we measured GFR as urinary clearance using continuous infusion of 125I-iothalamate. Kidney function reserve capacity was determined as increase in measured GFR after adding a dopamine infusion of 4.4-6 mg/h. Potential kidney donors were used as healthy controls and matched by age and sex to patients with ADPKD for comparisons across age groups and CKD stages. Hyperfiltration was defined by a loss of kidney function reserve capacity compared with healthy controls. RESULTS: A total of 300 participants were studied. In the youngest age group (18-29 years), measured GFR was not different between patients with ADPKD and healthy controls (103±21 versus 111±9 ml/min per 1.73 m2; P=0.14). In this age group kidney function reserve capacity was higher compared with healthy controls (11.1%±8.3% versus 5.3%±6.5%; P=0.04). Moreover, kidney function reserve capacity was similar to healthy controls in patients with ADPKD with early-stage disease (eGFR≥60 ml/min per 1.73 m2), either overall or when divided into fast or slow progressors according to their Mayo height-adjusted total kidney volume class. However, in patients with ADPKD, lower measured GFR was associated with lower kidney function reserve capacity (ß=1.0 [95% confidence interval, 0.5 to 1.5] % per 10 ml/min per 1.73 m2; P<0.001). Kidney function reserve capacity was therefore lower compared with healthy controls at older age and later CKD stages. CONCLUSIONS: Patients with early-stage ADPKD, either classified as having rapidly or slowly progressive disease, are able to increase their GFR in response to dopamine. Hyperfiltration, defined by a loss of kidney function reserve capacity, may therefore not be an early phenomenon in ADPKD.

The Effect of Tyrosine Kinase Inhibitors on the L-type Calcium Current in Rat Basilar Smooth Muscle Cells

OBJECTIVE: Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm. It has not yet been reported whether L-type Ca2+ channels play a role in tyrosine kinase inhibitors-induced vascular relaxation of cerebral artery. This study was undertaken to clarify the role of L-type Ca2+ channels in tyrosine kinase inhibitors-induced vascular relaxation, and to investigate the effect of tyrosine kinase inhibitors on L-type Ca2+ channels currents in freshly isolated smooth muscle cells from rat basilar artery. METHODS: The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recorded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. RESULTS: Patch clamp studies revealed a whole-cell current which resembles the L-type Ca2+ current reported by others. The amplitude of this current was decreased by nimodipine and increased by Bay K 8644. Genistein(n=5), tyrphostin A-23(n=3), A-25(n=6) 30micrometer reduced the amplitude of the L-type Ca2+ channel current in whole cell mode. In contrast, diadzein 30 micrometer (n=3), inactive analogue of genistein, did not decrease the amplitude of the L-type Ca2+ channels current. CONCLUSION: These results suggest that tyrosine kinase inhibitors such as genistein, tyrphostin A-23, A-25 may relax cerebral vessel through decreasing level of intracellular calcium, [Ca2+]i, by inhibition of L-type Ca2+ channel.

Effects of Tyrosine Kinase Inhibitors on the Calcium-Dependent K+ Current in Rat Basilar Smooth Muscle Cells

OBJECTIVE: Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm. It has not yet reported whether potassium channel plays a role in tyrosine kinase inhibitors-induced vascular relaxation of cerebral artery. This study is undertaken to clarify the role of potassium channel in tyrosine kinase inhibitors-induced vascular relaxation, and to investigate the effect of tyrosine kinase inhibitors on outward potassium currents in freshly isolated smooth muscle cells from rat basilar artery. METHODS: The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recoreded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. RESULTS: In present study, genistein(n=10), tyrphostin A-23(n=10), A-25(n=10) 30microM into bath solution increased the amplitude of the outward K+ current which was completely blocked by large conductance calcium-activated potassium channel(BK(Ca)) blocker, iberiotoxin(0.1microM), and calcium chelator, BAPTA, in whole cell mode. In contrast, diadzein 30microM(n=10), inactive analogue of genistein, did not increase the amplitude of the outward K+ current. CONCLUSION: Our results suggest tyrosine kinase inhibitors such as genistein, tyrphostin A-23 and A-25 increase the BK(Ca) channel activity in cerebral basilar smooth muscle cells, thereby contributing to the relaxation of cerebral artery.