Orientational variability of parallel arrays of cortical microtubules under the outer cell wall of the helianthus hypocotyl epidermis

The epidermis of Helianthus hypocotyl can be peeled off and, in the form of detached strips can be used as a model system to study the effect on cortical microtubule (cMTs) orientation of these factors, which are difficult to be manipulated in situ, such as apoplastic pH or applied stress. In the first step, however, the orientation and reorientation of cMTs in the epidermis in situ must be described. The cMTs under the epidermal wall in hypocotyl epidermis at different positions along the hypocotyl and on its opposite sides were studied by means of immunostaining, using epi-fluorescence microscopy. The angle l that parallel array of cMTs makes with cell longitudinal axis was measured. The variation of l in a population of cells was documented by l-histogram (frequency of cells exhibiting a particular l±Dl plotted against l value). The histograms were of either transverse type (maximum at l ~90°, denoted as type A) or oblique type (two maxima on both sides of the transverse direction, denoted as type B) in the apical part of the hypocotyl, and were either of B type or of longitudinal type (maximum at l ~0° or 180° denoted as type C) in the basal part. The change from A or B to C basipetally may be considered as due to the developmental trend in cMT orientation. The occurrence of B above A in some hypocotyls in their apical part strengthens the hypothesis on the autonomous reorientation of cMTs. The intermingled occurrence of Aand B reorientation in the upper part of hypocotyl is interpreted as amanifestation of a subtle control of cell growth in latitudinal direction. The majority of histograms were asymmetric showing predominance of cMT parallel arrays inclined as the middle part of the letter Z

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

AbstractCdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte

Autophagy inactivation in osteosarcoma leads to the appearance of poor prognosis-associated factors

Osteosarcoma (OS) is a bone cancer exhibiting a 20% survival rate for metastatic patients, which motivates the development of new therapeutic options. Among the various new treatment approaches, modulation of autophagy is the subject of rising interest. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. In OS, although the knockdown of key autophagy genes in human cell lines demonstrates a protumoral role of autophagy, the analysis of patient tumors indicates that lack of LC3-positive punctae at resection following neoadjuvant chemotherapy is a poor prognostic marker, suggesting that loss of autophagy is not detrimental for the tumor. In the present work, we analyzed the consequences of autophagy inactivation in OS cells both on tumor development and on bone microenvironment in an orthotopic syngeneic model. We found that inactivation of the autophagy-essential gene Atg5 in OS cells decreases their tumorigenic properties in vitro. However, these effects were no longer observed in vivo, likely due to microenvironment modifications such as overexpression of the major OS-promoting factor TGF-β or increased infiltration of Foxp3-positive and CD31-positive cells in Atg5 KO tumors. In addition, autophagy-deficient tumor cells stimulate the in vitro formation of osteoclast, the cells in charge of bone resorption which can release bone matrix-embedded growth factors thereby stimulating tumor growth. Taken together, these results suggest that Atg5 inactivation in OS cells is associated with microenvironment modifications known as poor prognosis-associated factors in OS, and could thus balance the negative cell-autonomous effects of autophagy suppression. Abbreviations: ACTB -β-actin; Atg -autophagy-related; Baf-A1 - Bafilomycin-A1; CSC -cancer stem cells; Col1A -type 1a collagen; d -day; HBSS -Hank’s balanced salt solution; LC3 -microtubule-associated protein 1 light chain 3 protein; SQSTM1/p62 -sequestosome; OB -osteoblast; OC -osteoclast; OS -osteosarcoma; TEM -transmission electron microscopy; TGF-β -transforming growth factor β; TRAP -acid phosphatase 5, tartrate-resistant