Lasp-1 Regulates Podosome Function

Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins

Common MicroRNA Signatures in Cardiac Hypertrophic and Atrophic Remodeling Induced by Changes in Hemodynamic Load

BACKGROUND: Mechanical overload leads to cardiac hypertrophy and mechanical unloading to cardiac atrophy. Both conditions produce similar transcriptional changes including a re-expression of fetal genes, despite obvious differences in phenotype. MicroRNAs (miRNAs) are discussed as superordinate regulators of global gene networks acting mainly at the translational level. Here, we hypothesized that defined sets of miRNAs may determine the direction of cardiomyocyte plasticity responses. METHODOLOGY/PRINCIPAL FINDINGS: We employed ascending aortic stenosis (AS) and heterotopic heart transplantation (HTX) in syngenic Lewis rats to induce mechanical overloading and unloading, respectively. Heart weight was 26±3% higher in AS (n = 7) and 33±2% lower in HTX (n = 7) as compared to sham-operated (n = 6) and healthy controls (n = 7). Small RNAs were enriched from the left ventricles and subjected to quantitative stem-loop specific RT-PCR targeting a panel of 351 miRNAs. In total, 153 miRNAs could be unambiguously detected. Out of 72 miRNAs previously implicated in the cardiovascular system, 40 miRNAs were regulated in AS and/or HTX. Overall, HTX displayed a slightly broader activation pattern for moderately regulated miRNAs. Surprisingly, however, the regulation of individual miRNA expression was strikingly similar in direction and amplitude in AS and HTX with no miRNA being regulated in opposite direction. In contrast, fetal hearts from Lewis rats at embryonic day 18 exhibited an entirely different miRNA expression pattern. CONCLUSIONS: Taken together, our findings demonstrate that opposite changes in cardiac workload induce a common miRNA expression pattern which is markedly different from the fetal miRNA expression pattern. The direction of postnatal adaptive cardiac growth does, therefore, not appear to be determined at the level of single miRNAs or a specific set of miRNAs. Moreover, miRNAs themselves are not reprogrammed to a fetal program in response to changes in hemodynamic load

How Reliable Is Ki-67 Immunohistochemistry in Grade 2 Breast Carcinomas? A QA Study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas

Podosomes in space

International audienceMigration of macrophages is a key process for a variety of physiological functions, such as pathogen clearance or tissue homeostasis. However, it can also be part of pathological scenarios, as in the case of tumor-associated macrophages. This review presents an overview of the different migration modes macrophages can adopt, depending on the physical and chemical properties of specific environments, and the constraints they impose upon cells. We discuss the importance of these environmental and also of cellular parameters, as well as their relative impact on macrophage migration and on the formation of matrix-lytic podosomes in 2D and 3D. Moreover, we present an overview of routinely used and also newly developed assays for the study of macrophage migration in both 2D and 3D contexts, their respective advantages and limitations, and also their potential to reliably mimic in vivo situations

Tailored melt electrowritten scaffolds for the generation of sheet-like tissue constructs from multicellular spheroids

Melt electrowriting (MEW) is an additive manufacturing technology that is recently used to fabricate voluminous scaffolds for biomedical applications. In this study, MEW is adapted for the seeding of multicellular spheroids, which permits the easy handling as a single sheet-like tissue-scaffold construct. Spheroids are made from adipose-derived stromal cells (ASCs). Poly(ε-caprolactone) is processed via MEW into scaffolds with box-structured pores, readily tailorable to spheroid size, using 13-15 µm diameter fibers. Two 7-8 µm diameter "catching fibers" near the bottom of the scaffold are threaded through each pore (360 and 380 µm) to prevent loss of spheroids during seeding. Cell viability remains high during the two week culture period, while the differentiation of ASCs into the adipogenic lineage is induced. Subsequent sectioning and staining of the spheroid-scaffold construct can be readily performed and accumulated lipid droplets are observed, while up regulation of molecular markers associated with successful differentiation is demonstrated. Tailoring MEW scaffolds with pores allows the simultaneous seeding of high numbers of spheroids at a time into a construct that can be handled in culture and may be readily transferred to other sites for use as implants or tissue models

Lasp-1 is a component of the podosome ring structure.

<p>(<b>A, B</b>) Confocal micrographs of primary human macrophages transfected with constructs encoding (A) EGFP-Lasp-1 and (B) EYFP-Vinculin (green), respectively, and stained with vinculin- or Lasp-1-specific antibodies (with Alexa488- or Alexa 568-conjugated secondary antibody) for endogenous vinculin or Lasp-1, respectively, and Cy5-conjugated phalloidin for F-actin (blue). White box indicates detail images below. Bars represent 10 µm. (<b>C, D</b>) 3D reconstruction of single podosomes of primary human macrophages. Left panels: xyz mode, right panels: xzy mode for the same podosome. (C) Cells were transfected with EGFP-Lasp-1 and stained with Alexa568-phalloidin for F-actin (podosome core) or vinculin-specific antibody (with Alexa568-labeled secondary antibody) for vinculin (podosome ring structure; red). (D) Both untransfected cells (stained with Alexa488-phalloidin for F-actin; green) and cells transfected with EYFP-vinculin (green) were stained with Lasp-1-specific antibody (with Alexa568-labeled secondary antibody) for endogenous Lasp-1 (red). (<b>E</b>) Confocal micrographs of a rat smooth muscle cell (A7r5), stained with Alexa-594 phalloidin for F-actin (red, upper panel) or zyxin-specific antibody (with Alexa594-labeled secondary antibody) for endogenous zyxin (red, lower panel) and co-stained with Lasp-1-specific antibody (with Alexa488-labeled secondary antibody) for endogenous Lasp-1 (green). Lasp-1 and its binding partner zyxin colocalize at F-actin-rich podosomes (merge) in PDBu-treated A7r5 cells. Bars represent 25 µm.</p

Podosome lifetime is altered in Lasp-1 knockdown macrophages.

<p>Primary human macrophages were treated with Lasp-1-specific siRNA (Oligo A or C) or control siRNA. (<b>A</b>) Examples for podosome dynamics included in the analysis. Shown are single podosomes of cells co-transfected with siRNA and Lifeact-TagGFP2. Podosome lifetime is defined as 1) appearance to dissolution, 2) appearance to fission, or 3) fusion to dissolution. (<b>B–D</b>) Measurement of podosome lifetime from cells treated with Lasp-1-specific siRNA, compared to controls. Double asterisks indicate values highly significant different from controls with <i>P</i><0.004 (means+SD, n = 3×4) (B). (C, D) Graph showing detailed podosome lifetime analysis, data from (B), of cells transfected with Lasp-1-specific siRNA (Oligo A (C; purple); Oligo C (D; brown), compared to controls (C, D; grey; (means+SE; n = 3×4;)).</p

Lasp-1 mutants lacking either the LIM domain, the NEBU repeats and the Linker region, or the SH3 domain are still localized at podosomes.

<p>(<b>A</b>) Domain structure of Lasp-1 comprising an aminoterminal LIM domain, two nebulin (NEBU) repeats and an adjacent Linker region mediating actin binding, and a carboxyterminal SH3 (Src homology 3) domain that binds zyxin and palladin (N = aminoterminus, C = carboxyterminus). (<b>B</b>) Confocal micrographs of primary human macrophages transfected with EGFP-Lasp-1 deletion constructs (green) and stained with vinculin-specific antibody (with Alexa568-conjugated secondary antibody) for vinculin (red) and Cy5- phalloidin for F-actin. Constructs include EGFP-Lasp-1 wildtype (wt), EGFP-Lasp-1ΔLIM (lacking the LIM domain), EGFP-Lasp-1ΔSH3 (lacking the SH3 domain), and EGFP-Lasp-1ΔN-L (lacking the NEBU repeats and the Linker region). White boxes indicate detail images below. Bars represent 10 µm.</p