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mUBPY, endocytic vesicle traffic and microtubule mediated transport in acrosome biogenesis. Comparative study between wild-type and Wobbler (L967Q Vps54) mice
No full textAcrosome biogenesis is a multistep process, essential for fertilization, involving poorly understood events, like vesicular trafficking and microtubular transport. We found that the deubiquitinating enzyme UBPy, in mouse, marks step-by-step the development of the acrosomal vesicle. Recently by studies on transfected cells, UBPy is emerging to play a key and no redundant role in endosomal sorting, resulting to be fundamental for the cell life. In this work we studied in male germ cells the endosomal trafficking machinery, in particular we detected the early endosome compartment and the ESCRT-0 complex, interestingly mUBPy colocalizes and interacts with Hrs and Hbp (Hrs-binding protein) at the early endosomes. We investigated also Vps54, a component of the Golgi associated retrograde protein complex which is involved in vesicles transport from early endosomes to trans Golgi network. Altogether our findings demonstred for the first time the presence of a mutiprotein complex in spermatogenic cells. Interestingly, proteins belonging to vesicular compartment participate and cooperate with UBPy to the formation of the acrosome, and here remain in the mature sperm, strongly suggesting that the endosome system, through an UBPy-microtubular manchette-mediated transport, could contribute to the biogenesis of the acrosome. The missense mutation L967Q in Vps54 marks the wobbler mouse phenotype. Wobbler mouse is an animal model for motor neuron neurodegenerative disorders with associated male sterility. We provided the first characterization of wobbler reproductive apparatus, detecting the absence of the acrosome, an abnormal shape of sperm s head and a defective vesicular transport
USP8, a regulator of endosomal sorting, is involved in mouse acrosome biogenesis through interaction with the spermatid ESCRT-0 complex and microtubules
No full textUbiquitin-specific peptidase 8 (USP8) is a deubiquitinating
enzyme that works as a regulator of endosomal sorting and
vesicle morphology in cultured cells. Its function in vivo is,
however, unknown as USP8 gene deletion leads to embryonic
lethality. Previously, we have shown that USP8 is highly
expressed in male germ cells. These cells develop a peculiar
acidic vesicle that is indispensable for fertilization, the acrosome;
USP8 might be involved in vivo in acrosomogenesis. The
objective of this study was to test this hypothesis by determining
if selective components of the early endosomal machinery
interact functionally with USP8 during acrosomogenesis using
protein-protein interaction assays and double/triple immunolabeling.
Moreover, by exploiting the characteristic of USP8 that
exhibits a microtubule interacting and trafficking/transport
(MIT) domain, we verified whether USP8 effectively associates
with spermatid microtubules by microtubule cosedimentation
and binding assays. USP8 was able to interact with spermatid
ESCRT-0 (endosomal-sorting complex required for transport-0)
and microtubule structures; USP8/ESCRT-0-labeled vesicles,
monitored by fluorescence microscopy, were found to contribute
to acrosome formation while USP8 can directly link, via its
MIT domain, the labeled vesicles/developing acrosome to
microtubules, which could favor both acrosome assembly and
shaping. VPS54, the vacuolar-sorting protein responsible for
early endocytic retrograde transport, was here detected for the
first time in male germ cells; VPS54 followed the intracellular
route of USP8/ESCRT-0-labeled vesicles during acrosomogenesis.
We concluded that in vivo USP8 has a role strongly
associated with acrosome biogenesis and that the early
endosome pathway is significantly involved in the process,
which suggests that the acrosome could be a novel lysosomerelated
organelle
cAMP-Epac2-mediated activation of Rap1 in developing male germ cells : RA-RhoGAP as a possible direct down-stream effector
No full textRap1 is a small GTPase that functions as a positional signal and organizer of cell architecture. Recently Rap1 is emerged to play a critical role during sperm differentiation since its inactivation in haploid cells leads to a premature release of spermatids from the supporting Sertoli cell resulting in male infertility. How Rap1 is activated in spermatogenic cells has not yet been determined. Our objective was to investigate on a possible cAMP-mediated activation of Rap1 employing a cAMP analogue selective to Epac, the Rap1 activator directly responsive to cAMP, for stimulating cultured testis germ cells. Here we provide biochemical, cellular and functional evidence that the Epac variant known as Epac2 is expressed as both a transcript and a protein and that it is able to promote Rap1 activation in the cultured cells. A time course immunofluorescence analysis carried out on stimulated cells revealed the translocation of endogenous Epac2, which is cytosolic, towards the site where Rap1 is located, i.e., the Golgi complex, thus documenting the effective Rap1-Epac2 protein interaction 'in vivo' leading to Rap1-GTP loading. A combination of biochemical and molecular techniques supported the immunofluorescence data. The search for the presence of a putative Rap1 downstream effector, described in differentiating somatic cells as a target of cAMP-Epac-activated Rap1, revealed the presence in spermatogenic cells of RA-RhoGAP, a Rap1-activated Rho GTPase-activating protein. Taken together, our results, obtained with endogenously expressed proteins, are consistent with a cAMP/Epac2/Rap1-mediated signaling that could exert its action, among others, through RA-RhoGAP to promote the progression of spermatogenesis
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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