Campos, R. | Search bioRxiv

bioRxiv2023

Radial askew endodermal cell divisions reveal IRK functions in division orientation

Oriented cell divisions establish plant tissue and organ patterning and produce different cell types; this is particularly true of the highly organized Arabidopsis root meristem. Mutant alleles of INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) exhibit excess cell divisions in the root endodermis. IRK is a transmembrane receptor kinase that localizes to the outer polar domain of these cells, which suggests directional signal perception is necessary to repress endodermal cell division. Here, a detailed examination revealed many of the excess endodermal divisions in irk have division planes that specifically skew towards the outer lateral side, therefore we termed them radial askew divisions. Expression of an IRK truncation, lacking the kinase domain, retains polar localization and rescues these radial askew divisions, but the roots exhibit excess periclinal endodermal divisions. Using markers of cell identity, we show that the daughters of radial askew divisions transition from endodermal to cortex identity similar to those of periclinal divisions. These results extend the requirement for IRK beyond repression of cell division activity to include cell division plane positioning. Based on its polarity, we propose that IRK at the outer lateral endodermal cell face participates in division plane positioning to ensure normal root ground tissue patterning.

plant biology↗

bioRxiv2018

A freeze-and-thaw induced-fragment of the microtubule-associated protein Tau in rat brain extracts: implications for the study of neurodegenerative diseases

Tau is a microtubule-associated protein responsible for controlling the stabilization of microtubules in neurons. Tau function is regulated by phosphorylation. However, in some neurological diseases Tau becomes aberrantly hyperphosphorylated, which contributes to the pathogenesis of neurological diseases, known as tauopathies. Western blotting (WB) has been widely employed to determine Tau levels in neurological disease models. However, Tau quantification by WB should be interpreted with care, as this approach has been recognized as prone to produce artifactual results if not properly performed. In this study, our goal was to evaluate the influence of a freeze-and-thaw cycle, a common procedure preceding WB, to the integrity of Tau in brain homogenates from rats, 3xTg-AD mice and human samples. Homogenates were prepared in ice-cold RIPA buffer supplemented with protease/phosphatase inhibitors. Immediately after centrifugation, an aliquot of the extracts was analyzed via WB to quantify total and phosphorylated Tau levels. The remaining aliquots were stored for at least 2 weeks at either -20{degrees}C or -80{degrees}C and then subjected to WB. Extracts from rodent brains submitted to freeze-and-thaw presented a ~25 kDa fragment immunoreactive to anti-Tau antibodies. An in-gel digestion followed by mass spectrometry analysis in excised bands revealed this ~25 kDa species corresponds to a Tau fragment. Freeze-and-thaw-induced Tau proteolysis was detected even when extracts were stored at -80{degrees}C. This phenomenon was not observed in human samples at any storage condition tested. Based on these findings, we strongly recommend the use of fresh extracts of brain samples in molecular analysis of Tau levels in rodents.

biochemistry↗