LINCing the nuclear envelope to gametogenesis.

Gametogenesis combines two important features: reduction of the genome content from diploid to haploid by carefully partitioning chromosomes, and the subsequent differentiation into fertilization-competent gametes, which in males is characterized by profound nuclear restructuring. These are quite difficult tasks and require a tight coordination of different cellular mechanisms. Recent studies in the field established a key role for LINC complexes in both meiosis and sperm head formation. LINC complexes comprise SUN and KASH domain proteins that form nuclear envelope (NE) bridges, linking the nucleoskeleton to the cytoskeleton. They are well known for their crucial roles in diverse cellular and developmental processes, such as nuclear positioning and cell polarization. In this review, we highlight key roles ascribed to LINC complexes and to the nucleocytoskeletal connection in gametogenesis. First, we give a short overview about the general features of LINC components and the profound reorganization of the NE in germ cells. We then focus on specific roles of LINC complexes in meiotic chromosome dynamics and their impact on pairing, synapsis, and recombination. Finally, we provide an update of the mechanisms controlling sperm head formation and discuss the role of sperm-specific LINC complexes in nuclear shaping and their relation to specialized cytoskeletal structures that form concurrently with nuclear restructuring and sperm elongation.

Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms.

Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, expression and developmental regulation of myofilament proteins of mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues using a lacZ reporter gene driven by a cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images, and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in precise synchrony with that in the heart. However, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically pre-set developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart.

The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus.

Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye.

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.

At the crossroads of SUMO and NF-kappaB.

BACKGROUND: Recognition of pathogens by immune receptors leads to activation of macrophages, dendritic cells, and lymphocytes. Signals are communicated to enhance expression of target molecules such as cytokines and adhesion molecules, depending on activation of various inducible transcription factors, among which the family NF-kappaB transcription factors plays an evolutionarily conserved and critical role. Classical activation of NF-kappaB involves phosphorylation, polyubiquitination and subsequent degradation of the inhibitor molecules of NF-kappaB, referred to as IkappaB. Modification of IkappaBalpha, one of the mammalian IkappaB isoforms, with the small ubiquitin-like modifier (SUMO) results its protection from degradation. PRESENTATION OF THE HYPOTHESIS: SUMO-IkappaBalpha localizes in the nucleus. The nuclear SUMO-IkappaBalpha pool may be dynamic. SUMO-IkappaBalpha functions as synergy control factor. TESTING THE HYPOTHESIS: Immunoprecipitation from cellular fractions, 35S methionine pulse-chase, and FRET assays should reveal the localization of SUMO-IkappaBalpha and the dynamics of the pool. Expression of SUMOylation defective IkappaBalpha in an IkappaBalpha -/- background should yield insights into the function of SUMO-IkappaBalpha. IMPLICATION OF THE HYPOTHESIS: IkappaBalpha contains the required SUMOylation motif but IkappaBbeta does not. The suggested study would provide evidence whether or not IkappaBalpha and IkappaBbeta can substitute each other. In addition, the suggested assays would reveal a possible redundancy in controlling transcriptional activity of NF-kappaB.

TGFbeta1 signaling via alphaVbeta6 integrin.

BACKGROUND: Transforming growth factor beta1 (TGFbeta1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFbeta1 mediated growth inhibition, suggesting TGFbeta1 participation in the development of these cancers. The tumor suppressor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFbeta1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFbeta1 induced growth inhibition, thus requiring a SMAD4 independent TGFbeta1 pathway. RESULTS: Here we report that mature TGFbeta1 is a ligand for the integrin alphaVbeta6, independent of the common integrin binding sequence motif RGD. After TGFbeta1 binds to alphaVbeta6 integrin, different signaling proteins are activated in TGFbeta1-sensitive carcinoma cells, but not in cells that are insensitive to TGFbeta1. Among others, interaction of TGFbeta1 with the alphaVbeta6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells. CONCLUSIONS: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.