Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin

Oocytes of the leopard frog, Rana pipiens, contain soluble tubulin which was previously shown to exist predominantly in megadalton (MDa) fractions and that fails to readily assemble in vitro. In order to further characterize these tubulin complexes, DEAE Sepharose chromatography, Sephacryl S-300 size exclusion columns and specific immunoprecipitation were used. The results revealed the presence of alpha-, beta-, and gamma-tubulin associated with several other proteins in the soluble fraction of Rana pipiens ovarian oocytes. These Rana oocyte tubulin complexes appear to be analogous to those recently reported in Xenopus ovulated eggs as gamma-tubulin ring complexes. This seems true since both size (estimates, i.e. approximately 2MDa) and protein components are similar. Furthermore, both alpha- and gamma-tubulin antibodies immunoprecipitated identical protein bands from Rana oocyte soluble fraction. These putative Rana gamma-tubulin ring proteins include 107, 97, 95, 90 and 75 kDa components which are similar in size to those found in Xenopus and other species. Rana appears to belong to a select group in which gamma-tubulin complexes contain significant alpha- and beta-tubulin (i.e., Xenopus and sheep), while other species such as Drosophila, Aspergillus, Saccharomyces, human cells and many other mammalian cells tested lack the other tubulin components. The heterogeneity in both size and protein components of Rana oocyte gamma-tubulin ring complexes may reflect different states of tubulin complex assembly. The lower vertebrate oocyte is hypothesized to act as a repository and prestaging point for the assembly of gamma-tubulin ring complexes which will become the maternal contribution to the centrosomes of the embryo. While the gamma-tubulin ring complexes of vertebrate eggs have been described previously, this is the first report biochemically characterizing soluble gamma-tubulin complexes in vertebrate ovarian oocytes.     

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Biochemical and cytological changes associated with expression of deregulated pp60src in Xenopus oocytes.

We have examined the effects of Xenopus pp60c-src with constitutive kinase activity on the morphology and maturation of Xenopus laevis oocytes. When RNA encoding this deregulated variant was injected into stage VI oocytes, we observed a gross alteration in the cortex of the oocyte. This alteration involved aggregation of pigment and invagination of the cortex in a large area proximal to the site of injection. This phenomenon was not seen in oocytes injected with RNA encoding wild-type pp60c-src. We have correlated this phenomenon with the tyrosine phosphorylation of 84- and 100-kDa proteins. These phosphorylated proteins colocalized with the alteration in the oocyte cortex when assayed by both biochemical and immunocytochemical methods. Neither the pigment aggregation nor phosphorylation of the 84- and 100-kDa proteins was observed in oocytes expressing a nonmyristoylated version of the deregulated pp60c-src. Expression of deregulated Xenopus fyn, a src-family member, resulted in a phenotype similar to that seen with deregulated src. However, in the fyn-injected oocytes, many more proteins were phosphorylated on tyrosine than in the src-injected oocytes. Progesterone stimulation of oocytes expressing deregulated pp60c-src resulted in an increase in the number of tyrosine-phosphorylated proteins. This change may represent the response of pp60src to the resumption of the cell cycle in maturing oocytes. These data suggest that the oocyte may be a particularly useful system for investigating the role of pp60c-src in the regulation of cytoskeletal structure and in the regulation of events associated with the cell cycle.     

Two distinct Staufen isoforms in Xenopus are vegetally localized during oogenesis

Localization of mRNA is an important way of generating early asymmetries in the developing embryo. In Drosophila, Staufen is intimately involved in the localization of maternally inherited mRNAs critical for cell fate determination in the embryo. We show that double-stranded RNA-binding Staufen proteins are present in the oocytes of a vertebrate, Xenopus, and are localized to the vegetal cytoplasm, a region where important mRNAs including VegT and Vg1 mRNA become localized. We identified two Staufen isoforms named XStau1 and XStau2, where XStau1 was found to be the principal Staufen protein in oocytes, eggs, and embryos, the levels of both proteins peaking during mid-oogenesis. In adults, Xenopus Staufens are principally expressed in ovary and testis. XStau1 was detectable throughout the oocyte cytoplasm by immunofluorescence and was concentrated in the vegetal cortical region from stage II onward. It showed partial codistribution with subcortical endoplasmic reticulum (ER), raising the possibility that Staufen may anchor mRNAs to specific ER-rich domains. We further showed that XStau proteins are transiently phosphorylated by the MAPK pathway during meiotic maturation, a period during which RNAs such as Vg1 RNA are released from their tight localization at the vegetal cortex. These findings provide evidence that Staufen proteins are involved in targeting and/or anchoring of maternal determinants to the vegetal cortex of the oocyte in Xenopus. The Xenopus oocyte should thus provide a valuable system to dissect the role of Staufen proteins in RNA localization and vertebrate development.     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.     

Immunological identification of the karyophilic, histone-binding proteins N1 and N2 in somatic cells and oocytes of diverse amphibia

A polypeptide pair designated N1/N2 (Mr 100 000 and 110 000) is an exceptionally acidic and abundant nuclear protein of oocytes of the toad, Xenopus laevis, and is characterized by a pronounced karyophilia. These proteins have been shown to form specific complexes with free, i.e., non-chromatin-bound histones H3 and H4 (Kleinschmidt & Franke, Cell 29 (1982) 799) [3]. In order to study these proteins and their possible counterparts in other species, antibodies were produced in guinea pigs against proteins N1/N2 purified from Xenopus oocyte nuclei. Using gel electrophoresis, peptide map analysis, immunoblotting techniques and immuno fluorescence microscopy the existence of polypeptides identical in Mr value and charge to polypeptide N1 of oocytes was demonstrated in cultured somatic cells of Xenopus laevis, where it was also highly enriched in cell nuclei, although the cellular concentration was much lower than in oocytes. A similar, if not identical protein, was recognized in nuclei of diverse other cell types including hepatocytes, enterocytes, ovarian follicle cells, and Sertoli cells of testis, of Xenopus, Rana temporaria, R. esculenta, Pleurodeles waltlii but not in erythrocytes and later stages of spermiogenesis. When nuclear proteins from oocytes of different amphibian species were examined with these antibodies it was found that the Mr values of N1/N2 proteins were considerably different in different species, ranging from Mr 110 000 to 190 000. Immunoprecipitation and gel electrophoretic analysis under non-denaturing conditions showed that a significant proportion of these proteins was contained in complexes with histones H3 and H4. The results demonstrate that proteins N1/N2 are not special proteins of oocytes of Xenopus laevis but occur in various other cells of diverse amphibian species. The widespread occurrence of these karyophilic proteins indicates that at least one function of these proteins, i.e., selective binding of the arginine-rich histones H3 and H4, is not exclusive to oocytes but may also contribute to the regulation of histone pools and chromatin formation in other cell types.     

Inositol 1,4,5-trisphosphate receptor associated with focal contact cytoskeletal proteins

The linkage between inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and cytoskeletal proteins is considered to be important in cell function. In the present study, the association of IP(3)R subtypes with cytoskeletal proteins was examined using monoclonal antibodies specific to each IP(3)R subtype. We found that IP(3)R type 2 colocalized with talin, a focal contact cytoskeletal protein. IP(3)R type 2 exhibited a patchy distribution in the peripheral cytoplasm differently from type 1 and type 3 IP(3)R. Furthermore, IP(3)R subtypes co-immunoprecipitated with talin, vinculin and alpha-actin, but not alpha-actinin or paxillin.     

Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.     

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.     

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.     

Rous sarcoma virus-transformed fibroblasts and cells of monocytic origin display a peculiar dot-like organization of cytoskeletal proteins involved in microfilament-membrane interactions

By immunofluorescence and interference reflection microscopy (IRM) we found that F-actin and a group of cytoskeletal proteins involved in microfilament-membrane interaction, including vinculin, alpha-actinin, fimbrin and talin, are specifically organized in discrete dot-like structures corresponding to cell-substratum contact sites in both monocytes and monocyte-derived cells such as macrophages and osteoclasts. These proteins have a precise topological distribution; vinculin and talin form a doughnut-like ring, while actin, fimbrin and alpha-actinin are organized in dots matching the rings. An identical dot-like organization of F-actin and associated cytoskeletal proteins was also detected in malignant fibroblasts transformed by Rous Sarcoma virus (RSV) but not in the corresponding untransformed cells in culture. It is concluded that RSV transformation induces fibroblasts to express a cytoskeletal organization and a pattern of adhesion that are normally found in cells of monocytic origin. We propose that the occurrence of this cytoskeletal organization in RSV-transformed fibroblasts and in monocyte-derived cells may reflect a common ability to migrate across anatomical boundaries.     

Xenopus eggs express an identical DNA methyltransferase, Dnmt1, to somatic cells

In mouse, an oocyte-specific short isoform of DNA methyltransferase-1 (Dnmt1) lacking amino terminal 118 amino acid residues exists and plays a crucial role in maintaining the methylation state of imprinted genes during early embryogenesis [Howell et al. (2001) Cell 104, 829-838]. To address the question of whether or not Xenopus oocyte expresses such a short isoform, we raised monoclonal antibodies against the amino-terminal portion of Xenopus Dnmt1. Two of the isolated monoclonal antibodies, 3C6 and 4A8, were determined to recognize (1-32) and (115-126) of Xenopus Dnmt1, respectively. The amounts of Dnmt1 in Xenopus eggs were determined to be similar, 10.0 2.5, 8.0 0.8, and 8.2 0.2 ng per egg with monoclonal antibodies 3C6 and 4A8, and polyclonal antibodies, respectively. This indicated that Dnmt1 in Xenopus mature eggs had an identical amino-terminal sequence to the amino acid sequence deduced from the cDNA. Together with the fact that Dnmt1 in A6 cells immuno-reacted with all the monoclonal antibodies isolated and with the polyclonal antibodies, we concluded that Dnmt1 expressed in Xenopus mature eggs possesses an identical amino-terminal sequence to that in somatic cells. Immuno-purified Xenopus Dnmt1 in mature eggs showed similar specific activity to that in proliferating A6 cells and that of mouse recombinant Dnmt1.     

Structure and function of the human oocyte-cumulus-corona cell complex before and after ovulation

Summary The fine structure of the human cumulus oophorus has been reviewed on the basis of scanning and transmission electron microscopic observations as well as of immunofluorescence data. Tissues sampled from preovulatory ovarian follicles and cumulus-enclosed oocytes and fertilized eggs (collected from the oviduct or obtained during in vitro fertilization procedures) have been evaluated from a microtopographic and morphodynamic point of view in order to better clarify the possible role of this population of cells. In particular, the following aspects have been studied and discussed: the presence of multiple close contacts (modulated by the interposition of the zona pellucida) between the oocyte surface and the long microvillous evaginations projecting from the inner aspect of corona cells surface (through these structures the intraovarian cumulus oophorus may control oocyte growth and metabolism up until the time of ovulation); the occurrence of different subpopulations of cells (steroid-synthetic cells, cells producing adhesive proteins, leukocytes, macrophages) in the postovulatory, extraovarian cumulus oophorus surrounding oocytes, zygotes and early developing embryos. All these elements found in the cumulus mass may positively act, through their paracrine activities, on the chemical composition of the microenvironment in which fertilization occurs.     

Tension development during contractile stimulation of smooth muscle requires recruitment of paxillin and vinculin to the membrane

Cytoskeletal reorganization of the smooth muscle cell in response to contractile stimulation may be an important fundamental process in regulation of tension development. We used confocal microscopy to analyze the effects of cholinergic stimulation on localization of the cytoskeletal proteins vinculin, paxillin, talin and focal adhesion kinase (FAK) in freshly dissociated tracheal smooth muscle cells. All four proteins were localized at the membrane and throughout the cytoplasm of unstimulated cells, but their concentration at the membrane was greater in acetylcholine (ACh)-stimulated cells. Antisense oligonucleotides were introduced into tracheal smooth muscle tissues to deplete paxillin protein, which also inhibited contraction in response to ACh. In cells dissociated from paxillin-depleted muscle tissues, redistribution of vinculin to the membrane in response to ACh was prevented, but redistribution of FAK and talin was not inhibited. Muscle tissues were transfected with plasmids encoding a paxillin mutant containing a deletion of the LIM3 domain (paxillin LIM3 dl 444–494), the primary determinant for targeting paxillin to focal adhesions. Expression of paxillin LIM3 dl in muscle tissues also inhibited contractile force and prevented cellular redistribution of paxillin and vinculin to the membrane in response to ACh, but paxillin LIM3 dl did not inhibit increases in intracellular Ca²⁺ or myosin light chain phosphorylation. Our results demonstrate that recruitment of paxillin and vinculin to smooth muscle membrane is necessary for tension development and that recruitment of vinculin to the membrane is regulated by paxillin. Vinculin and paxillin may participate in regulating the formation of linkages between the cytoskeleton and integrin proteins that mediate tension transmission between the contractile apparatus and the extracellular matrix during smooth muscle contraction. tissue transfection; plasmids; cytoskeleton; talin; immunofluorescence     

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Cytokeratin intermediate filament organisation and dynamics in the vegetal cortex of living Xenopus laevis oocytes and eggs

Cytokeratin intermediate filaments are prominent constituents of developing Xenopus oocytes and eggs, forming radial and cortical networks. In order to investigate the dynamics of the cortical cytokeratin network, we expressed EGFP-tagged Xenopus cytokeratin 1(8) in oocytes and eggs. The EGFP-cytokeratin co-assembled with endogenous partner cytokeratin proteins to form fluorescent filaments. Using time-lapse confocal microscopy, cytokeratin filament assembly was monitored in live Xenopus oocytes at different stages of oogenesis, and in the artificially-activated mature egg during the first cell cycle. In stage III to V oocytes, cytokeratin proteins formed a loose cortical geodesic network, which became more tightly bundled in stage VI oocytes. Maturation of oocytes into metaphase II-arrested eggs induced disassembly of the EGFP-cytokeratin network. Imaging live eggs after artificial activation allowed us to observe the reassembly of cytokeratin filaments in the vegetal cortex. The earliest observable structures were loose foci, which then extended into curly filament bundles. The position and orientation of these bundles altered with time, suggesting that forces were acting upon them. During cortical rotation, the cytokeratin network realigned into a parallel array that translocated in a directed manner at 5 microm/minute, relative to stationary cortex. The cytokeratin filaments are, therefore, moving in association with the bulk cytoplasm of the egg, suggesting that they may provide a structural role at the moving interface between cortex and cytoplasm.