Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/ immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation

Subcellular Localization of SUN2 Is Regulated by Lamin A and Rab5

SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved

CLIC4 (chloride intracellular channel 4)

Review on CLIC4 (chloride intracellular channel 4), with data on DNA, on the protein encoded, and where the gene is implicated

Abstract 1418: CLIC4 regulates carcinogenesis in a TGF-β context-dependent manner.

Abstract CLIC4 is a 28kD, ubiquitously expressed, redox-regulated, multifunctional protein. It is dimorphic and can transition between membrane bound or soluble forms in the cytoplasm. Cytoplasmic CLIC4 translocates to the nucleus in multiple cell types under conditions of metabolic stress and nuclear CLIC4 causes growth arrest, terminal differentiation and apoptosis. In vivo, CLIC4 is nuclear in quiescent epithelial cells with little stromal expression. In contrast, nuclear CLIC4 is lost from tumor epithelium and is highly upregulated in tumor stroma. We show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts of oncogenic ras transformed keratinocytes inhibits tumor growth, while elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth. These results identify CLIC4 as a tumor suppressor. We show that CLIC4 is an integral intermediate in TGF-β signaling, that overexpressing CLIC4 in tumor cell lines restores TGF-β mediated growth inhibition, and tumor cells in vivo overexpressing CLIC4 have enhanced TGF-β signaling. We have also analyzed the substantial upregulation of CLIC4 in tumor stroma. Reconstituting orthografts of mammary or squamous tumors with stromal cells overexpressing CLIC4 enhances tumor growth.Correspondingly, tumor growth is significantly inhibited in orthografts of tumor cells to hosts that lack stromal CLIC4. CLIC4 expression is increased in stromal cells by conditioned medium from tumor cells in a TGF-β dependent manner. In stromal cells genetically deleted of CLIC4, the conversion of fibroblasts to cancer associated myofibroblasts by TGF-β through p38 activation is prevented. CLIC4 is essential for preventing the de-activation of p38 by its phosphatase PPM1a. Stromal cells that overexpress CLIC4 enhance tumor cell invasion and EMT in vitro. Thus CLIC4, like TGF-β, has context dependent dual influence on tumor cell growth and progression. CLIC4 is an attractive therapeutic target both in cancer stages where TGF-β signal augmentation or inhibition is required due to responses in separate tissue compartments. Targeting CLIC4 would also be a more specific approach in therapy that would mitigate some of the severe side effects of global targeting of the multifunctional TGF-β pathway. Citation Format: Anjali Shukla, Rebecca Edwards, Yihan Yang, Alexandra Hahn, VC Padmakumar, Andrew Ryscavage, Kwang S. Suh, Stuart H. Yuspa. CLIC4 regulates carcinogenesis in a TGF-β context-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1418. doi:10.1158/1538-7445.AM2013-1418</jats:p

Functional Analysis of LINC Complexes in the Skin

The genome in eukaryotic cells is encased by two intricate and interconnected concentric membranes, which together with the underlying nuclear lamina form the nuclear envelope (NE). Two fundamental macromolecular structures are embedded within the nuclear envelope: the nuclear pore (NPC) and the LINC complex. The former perforates the nucleus controlling biomolecule trafficking between the nucleoplasm and the cytoplasm, while the latter integrates the nucleus via the cytoskeleton to the extracellular matrix. LINC complex structural and functional integrity is of utmost importance for various fundamental cellular functions. Mechanical forces are relayed into the nuclear interior via the LINC complex, which controls lamina organization, chromosome dynamics, and genome organization and stability. Thus, LINC constituents play pivotal roles in cellular architecture including organelle positioning, cell movement, tissue assembly, organ homeostasis, and organismal aging. The LINC complex oligomeric core contains several multi-isomeric, multifunctional, and often tissue-specific proteins. Therefore, for a proper functional analysis, genetic mouse models are an invaluable resource. Herein, we focus on the LINC complex roles in the skin and describe methods that enable the successful isolation of primary embryonic fibroblast and newborn skin cells, which can be then investigated functionally in vitro

Cytoskeletal Configuration Modulates Mechanically Induced Changes in Mesenchymal Stem Cell Osteogenesis, Morphology, and Stiffness

Mesenchymal stem cells (MSC) responding to mechanical cues generated by physical activity is critical for skeletal development and remodeling. Here, we utilized low intensity vibrations (LIV) as a physiologically relevant mechanical signal and hypothesized that the confined cytoskeletal configuration imposed by 2D culture will enable human bone marrow MSCs (hBMSC) to respond more robustly when LIV is applied in-plane (horizontal-LIV) rather than out-of-plane (vertical-LIV). All LIV signals enhanced hBMSC proliferation, osteogenic differentiation, and upregulated genes associated with cytoskeletal structure. The cellular response was more pronounced at higher frequencies (100 Hz vs 30 Hz) and when applied in the horizontal plane. Horizontal but not vertical LIV realigned the cell cytoskeleton, culminating in increased cell stiffness. Our results show that applying very small oscillatory motions within the primary cell attachment plane, rather than perpendicular to it, amplifies the cell’s response to LIV, ostensibly facilitating a more effective transfer of intracellular forces. Transcriptional and structural changes in particular with horizontal LIV, together with the strong frequency dependency of the signal, emphasize the importance of intracellular cytoskeletal configuration in sensing and responding to high-frequency mechanical signals at low intensities