Persistent homological cell tracking technology

Abstract In this paper, we develop a cell tracking method based on persistent homological figure detection technology. We apply our tracking method to 9 different time-series cell images and extract several kinds of cell movements. Being able to analyze various images with a single method allows researchers to systematically understand and compare different tracking data. Persistent homological cell tracking technology’s 9 parameters all have clear meanings. Thus, researchers can decide the parameters not by black box trial-and-error but by the purpose of their analysis. We use model data with ground truth to see our method’s performance. We compare persistent homological figure detection and cell tracking technology with Image-Pro, sure-foreground in watershed method, and cell detection methods in previous studies. We see that there are some cases where Image-Pro’s tracking stops and requires manual plots, while our method does not require manual plots. We show that our technology includes sure-foreground and has more information, and can be applied to different types of data that previously needed different methods. We also show that our technology is powerful as a detection technology by applying the technology to 5 different types of cell images

Calpain 6 Is Involved in Microtubule Stabilization and Cytoskeletal Organization▿†

The calpains are a family of Ca2+-dependent cysteine proteases implicated in various biological processes. In this family, calpain 6 (Capn6) is unique in that it lacks the active-site cysteine residues requisite for protease activity. During the search for genes downstream of the endothelin 1 (ET-1) signaling in pharyngeal-arch development, we identified Capn6. After confirming that the expression of Capn6 in pharyngeal arches is downregulated in ET-1-null embryos by in situ hybridization, we investigated its function. In Capn6-transfected cells, cytokinesis was retarded and was often aborted to yield multinucleated cells. Capn6 overexpression also caused the formation of microtubule bundles rich in acetylated α-tubulin and resistant to the depolymerizing activity of nocodazole. Green fluorescent protein-Capn6 overexpression, immunostaining for endogenous Capn6, and biochemical analysis demonstrated interaction between Capn6 and microtubules, which appeared to be mainly mediated by domain III. Furthermore, RNA interference-mediated Capn6 inactivation caused microtubule instability with a loss of acetylated α-tubulin and induced actin reorganization, resulting in lamellipodium formation with membrane ruffling. Taken together, these results indicate that Capn6 is a microtubule-stabilizing protein expressed in embryonic tissues that may be involved in the regulation of microtubule dynamics and cytoskeletal organization

A three-dimensional model with two-body interactions for endothelial cells in angiogenesis

Abstract We introduce a three-dimensional mathematical model for the dynamics of vascular endothelial cells during sprouting angiogenesis. Angiogenesis is the biological process by which new blood vessels form from existing ones. It has been the subject of numerous theoretical models. These models have successfully replicated various aspects of angiogenesis. Recent studies using particle-based models have highlighted the significant influence of cell shape on network formation, with elongated cells contributing to the formation of branching structures. While most mathematical models are two-dimensional, we aim to investigate whether ellipsoids also form branch-like structures and how their shape affects the pattern. In our model, the shape of a vascular endothelial cell is represented as a spheroid, and a discrete dynamical system is constructed based on the simple assumption of two-body interactions. Numerical simulations demonstrate that our model reproduces the patterns of elongation and branching observed in the early stages of angiogenesis. We show that the pattern formation of the cell population is strongly dependent on the cell shape. Finally, we demonstrate that our current mathematical model reproduces the cell behaviours, specifically cell-mixing, observed in sprouts

Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

<div><p>Calpains are Ca²⁺-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6's <i>in vivo</i> roles, we generated <i>Capn6</i>-deficient mice, in which a <i>lacZ</i> expression cassette was integrated into the <i>Capn6</i> gene. These <i>Capn6</i>-deficient mouse embryos expressed <i>lacZ</i> predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, <i>Capn6</i> expression was detected in skeletal myocytes, and <i>Capn6</i>-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, <i>Capn6</i>-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.</p></div

Coordinated linear and rotational movements of endothelial cells compartmentalized by VE-cadherin drive angiogenic sprouting

Summary: Angiogenesis is a sequential process to extend new blood vessels from preexisting ones by sprouting and branching. During angiogenesis, endothelial cells (ECs) exhibit inhomogeneous multicellular behaviors referred to as “cell mixing,” in which ECs repetitively exchange their relative positions, but the underlying mechanism remains elusive. Here we identified the coordinated linear and rotational movements potentiated by cell-cell contact as drivers of sprouting angiogenesis using in vitro and in silico approaches. VE-cadherin confers the coordinated linear motility that facilitated forward sprout elongation, although it is dispensable for rotational movement, which was synchronous without VE-cadherin. Mathematical modeling recapitulated the EC motility in the two-cell state and angiogenic morphogenesis with the effects of VE-cadherin-knockout. Finally, we found that VE-cadherin-dependent EC compartmentalization potentiated branch elongations, and confirmed this by mathematical simulation. Collectively, we propose a way to understand angiogenesis, based on unique EC behavioral properties that are partially dependent on VE-cadherin function

Increased differentiation of <i>Capn6</i>-deficient skm-primary cultured cells.

<p>(A) <i>Capn6</i> and <i>Gapdh</i> (internal control) mRNA was detected by PCR during the differentiation of skm-primary cultured cells. (B) Western blot analysis confirming the CAPN6 expression in skm-primary cultured cells. A band was detected around 70 kDa (indicated by arrow) in differentiating cells that was lost in <i>Capn6</i>-deficient cells. Thus, this 70-kDa band was presumed to be CAPN6, and all the other bands were considered to be non-specific. (C) Confocal images of skm-primary cultured cells. The cells were established from the skeletal muscles of 7-wk-old <i>Capn6^+/Y</i> and <i>Capn6^lacZ/Y</i> mice, and incubated in horse serum-containing medium for the indicated number of days to induce myotube differentiation. The nuclei and actin filaments of differentiated cells were visualized with DAPI (blue) and rhodamine-labeled phalloidin (red), respectively. Scale bar: 100 µm. (D) Average number of nuclei per cell in <i>Capn6^+/Y</i> and <i>Capn6^lacZ/Y</i> skm-primary cultured cells. Data are the mean values from three experiments (±s.e.m.), and 100 cells were counted in each experiment. The number of nuclei per cell was significantly greater in <i>Capn6^lacZ/Y</i> than in <i>Capn6^+/Y</i> after five days of differentiation (<i>Capn6^lacZ/Y</i> vs <i>Capn6^+/Y</i> are [2.16 [mean]±0.13 [s.e.m.] vs 1.44±0.04], [2.14±0.09 vs 1.75±0.04], and [2.14±0.11 vs 1.76±0.05] at 5, 7, and 9 days after induction, respectively). **, <i>P</i><0.01 by Student's t-test. Wt, <i>Capn6^+/Y</i> (♂); Hemi, <i>Capn6^lacZ/Y</i> (♂).</p

Advanced skeletal muscle development in <i>Capn6</i>-deficient embryos.

<p>(A–D) X-gal-stained coronal sections of the tongue of an E16.5 <i>Capn6^lacZ/+</i> (A, C) and <i>Capn6^lacZ/lacZ</i> (B, D) embryo. The boxed areas in A and B are enlarged in C and D, respectively. Scale bar: 50 µm. (E) The average diameter of tongue muscle fibers was measured in the anterior region (the areas in boxes a′ and b′ in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003668#pgen.1003668.s002" target="_blank">Figure S2</a>) of coronal sections. The average diameter was significantly larger in <i>Capn6^lacZ/lacZ</i> (9.13 [mean]±0.56 [s.e.m.] µm, n = 5) than in <i>Capn6^lacZ/+</i> (5.64±0.23 µm, n = 5). **, <i>P</i><0.01 by Student's t-test. (F) Western blot analysis of the right limbs of E14.5, E16.5, and E18.5 embryos using antibodies against desmin, α-sarcomeric actin, CAPN6, CAPN1, and β-actin. Blotting for β-actin served as an internal control. Wt, <i>Capn6^+/Y</i> (♂); Hemi, <i>Capn6^lacZ/Y</i> (♂); Het, <i>Capn6^lacZ/+</i> (♀); Ho, <i>Capn6^lacZ/lacZ</i> (♀).</p

Targeted disruption of the <i>Capn6</i> gene.

<p>(A) Schematic representation of the targeting strategy used to knock-in an <i>nls-lacZ</i> cassette into the mouse <i>Capn6</i> locus. Exons 1 to 3 are indicated by open boxes with exon numbers. The 5′- and 3′-probes for Southern blotting are shown as hatched boxes. PCR primer positions for genotyping are indicated by arrows (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003668#pgen.1003668.s004" target="_blank">Table S1</a> for primers). Neo^r, neomycin-resistance gene; pA, poly A tail; TK, thymidine kinase; A, <i>ApaI</i>; B, <i>BamHI</i>; E, <i>EcoRI</i>. (B) Southern blot (left) and PCR (right) analyses of genomic DNA extracted from mouse tails. The bands of the Southern blot represent <i>ApaI/BamHI</i>- or <i>EcoRI</i>-digested genomic DNA from wild-type and <i>Capn6^lacZ/Y</i> mice, probed with the 5′- or 3′-probe. (C–E) Western blot analyses confirming the absence of CAPN6 in <i>Capn6</i>-deficient mice. Total lysates were obtained from E10.5, E12.5, and E14.5 whole embryos (C), the tongue of E14.5, E16.5, and E18.5 embryos (D), and limb buds of E13.5 embryos (E). A band of around 70 kDa (indicated by arrows) was diminished in the <i>Capn6</i>-deficient embryos and was presumed to be CAPN6, and all the other bands were considered to be non-specific. Wt, <i>Capn6^+/Y</i> (♂); Hemi, <i>Capn6^lacZ/Y</i> (♂); Het, <i>Capn6^lacZ/+</i> (♀); Ho, <i>Capn6^lacZ/lacZ</i> (♀).</p