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HAX-1 is a multi-functional protein that is involved in the regulation of apoptosis, cell motility and calcium homeostasis. It is also reported to bind RNA: it associates with structural motifs present in the 3′ untranslated regions of at least two transcripts, but the functional significance of this binding remains unknown. Although HAX-1 has been detected in various cellular compartments, it is predominantly cytoplasmic. Our detailed localization studies of HAX-1 isoforms revealed partial nuclear localization, the extent of which depends on the protein isoform. Further studies demonstrated that HAX-1 is in fact a nucleocytoplasmic shuttling protein, dependent on the exportin 1 nuclear export receptor. Systematic mutagenesis allowed identification of the two nuclear export signals in the HAX-1 sequence. HAX-1 nuclear accumulation was observed after inhibition of nuclear export by leptomycin B, but also after specific cellular stress. The biological role of HAX-1 nuclear localization and shuttling remains to be established, but the HAX-1 transcript-binding properties suggest that it may be connected to mRNA processing and surveillance. In this study, HAX-1 status was shown to influence mRNA levels of DNA polymerase b, one of the HAX-1 mRNA targets, although this effect becomes pronounced only after specific stress is applied. Moreover, HAX-1 tethering to the reporter transcript caused a significant decrease in its expression. Additionally, the HAX-1 co-localization with P-body markers, reported here, implies a role in mRNA processing. These results suggest that HAX-1 may be involved in the regulation of expression of bound transcripts, possibly as part of the stress response. Structured digital abstract• HAX1 and DCP1A, colocalize by fluorescence microscopy (View Interaction: 1, 2) • HAX1 physically interacts with XPO1 by anti tagcoimmunoprecipitation (View interaction) Abbreviations CPEB, cytoplasmic polyadenylation element-binding protein; Crm1, chromosome region maintenance 1; Dcp1a, mRNA-decapping enzyme 1A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HuR, human antigen R, ELAV-like protein 1; LMB, leptomycin B; NES, nuclear export signal; POLB, DNA polymerase b; Pat1b, protein PAT1 homolog 1; rck/p54, ATP dependent RNA helicase DDX6; TG, thapsigargin; XPO1, exportin1.

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This chapter describes signaling pathways stimulated by the P2Y(2) nucleotide receptor (P2Y(2)R), that regulate cellular processes dependent on actin cytoskeleton dynamics in glioma C6 cells. P2Y(2)R coupled with G-proteins, in response to ATP or UTP, regulates the level of phosphatidylinositol-4,5-bisphosphate (PIP(2)) which modulates a variety of actin binding proteins and is involved in calcium response and activates Rac1 and RhoA proteins. The RhoA/ROCK signaling pathway plays an important role in contractile force generation needed for the assembly of stress fibers, focal adhesions and for tail retraction during cell migration. Blocking of this pathway by a specific Rho-kinase inhibitor induces changes in F-actin organization and cell shape and decreases the level of phosphorylated myosin II and cofilin. In glioma C6 cells these changes are reversed after UTP stimulation of P2Y(2)R. Signaling pathways responsible for this compensation are connected with calcium signaling. Stimulation of the Rac1 mediated pathway via G(o) proteins needs additional interaction between α(v)β(5) integrins and P2Y(2)Rs. Rac1 activation is necessary for cofilin phosphorylation as well as integrin activation needed for focal complexes formation and stabilization of lamellipodium. Inhibition of positive Rac1 regulation prevents glioma C6 cells from recovery of control cell like morphology.

HAX-1, a multifunctional protein involved in the regulation of apoptosis, cell migration, and calcium homeostasis, binds the 3¢ untranslated region motifs of specific transcripts. This suggests that HAX-1 plays a role in posttranscriptional regulation, at the level of mRNA stability/transport or translation. In this study, we analyze in detail HAX-1 colocalization with processing bodies (P-bodies) and its dependence on mRNA availability. Endogenous P-body markers DCP1 and Rck/p54 were shown to colocalize with endogenous HAX-1, but in case of the overexpressed proteins, only DCP1 displayed unperturbed colocalization with HAX-1. HAX-1 colocalization with DCP1 was observed in most of the cell lines studied, but its presence was not required for P-body formation, and its silencing caused an increase in P-body number. Preliminary mapping suggested that HAX-1 has more than one short P-body-targeting sequence. The pools of P-body-localized HAX-1 and cytosolic HAX-1 were demonstrated to dynamically exchange, suggesting steady flow of the protein. Active transcription was shown to be a factor in the localization of HAX-1 to P-bodies. Also, it was observed that HAX-1 localizes to some unidentified foci, which do not contain DCP1. In addition, it was demonstrated that HAX-1 status influences vimentin expression levels. Overall, HAX-1 was shown to colocalize with P-body markers and influence P-body number per cell in a manner dependent on mRNA availability. Presented data support the hypothesis that HAX-1 is involved in mRNA processing as an element of P-body interaction network.

Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y₂ receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y₂ stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.

Leaf senescence is a highly coordinated process which involves programmed cell death (PCD). Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are less well known. In this study, we correlated condensation of chromatin and nuclear DNA (nDNA) fragmentation, two main features of PCD during early senescence in barley leaves, with the appearance of nitric oxide (NO) within leaf tissue. With the help of the alkaline version of the comet assay, together with measurements of nDNA fluorescence intensity, we performed a detailed analysis of the degree of nDNA fragmentation. We localised NO in vivo and in situ within the leaf and photometrically measured its concentration with the NO-specific fluorochrome 4-amino-5-methylamino-2',7'-difluorofluorescein. We found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order: first nDNA fragmentation, in leaves of 6-day-old seedlings, and later chromatin condensation, in the apical part of leaves from 10-day-old seedlings. PCD did not start simultaneously even in neighbouring cells and probably did not proceed at the same rate. NO was localised in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. Localisation of NO in vascular tissue and in a large number of mesophyll cells during the senescence process might imply its transport to other parts of the leaf and its involvement in signalling between cells. The fact that the highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence and lower concentrations were found during later stages might suggest that NO plays an inductive role in PCD.

Ruch i migracja są jedną z głównych funkcji życiowych komórek. W odpowiedzi na różne bodźce, dynamiczny cytoszkielet aktynowy generuje siłę umożliwiającą komórce przemieszczanie się w trójwymiarowej sieci zewnątrzkomórkowej macierzy czy po płaskim podłożu. Wydłużanie filamentów aktynowych na ich kolczastych końcach wypycha błonę komórkową w kierunku migracji, formując strefę frontalną zwaną lamellipodium. Skurcz włókien naprężeniowych umożliwia oderwanie tylnej części komórki i przesunięcie jej do przodu. W odpowiedzi na bodźce ze środowiska, receptory komórki inicjują wiele szlaków sygnałowych powodujących reorganizację mikrofilamentów aktynowych oraz skurcz układu akto-miozynowego. Głównymi regulatorami tych procesów są białka z rodziny Rho, fosfolipidy PIP2 oraz jony wapnia. Receptory nukleotydowe P2Y2 w połączeniu z białkami G regulują poziom fosfatydyloinozytolu-4,5-bisfosforanu (PIP2), który moduluje funkcje białek wiążących aktynę i aktywuje białka Rac1 oraz RhoA. Szlak sygnałowy RhoA/ROCK odgrywa ważną rolę w generowaniu skurczu włókien naprężeniowych. Z kolei białko Rac1 poprzez swój efektor kinazę PAK1 reguluje procesy formujące lamellipodium oraz wysuwanie strefy wiodącej podczas migracji.