Actin cap associated focal adhesions and their distinct role in cellular mechanosensing

The ability for cells to sense and adapt to different physical microenvironments plays a critical role in development, immune responses, and cancer metastasis. Here we identify a small subset of focal adhesions that terminate fibers in the actin cap, a highly ordered filamentous actin structure that is anchored to the top of the nucleus by the LINC complexes; these differ from conventional focal adhesions in morphology, subcellular organization, movements, turnover dynamics, and response to biochemical stimuli. Actin cap associated focal adhesions (ACAFAs) dominate cell mechanosensing over a wide range of matrix stiffness, an ACAFA-specific function regulated by actomyosin contractility in the actin cap, while conventional focal adhesions are restrictively involved in mechanosensing for extremely soft substrates. These results establish the perinuclear actin cap and associated ACAFAs as major mediators of cellular mechanosensing and a critical element of the physical pathway that transduce mechanical cues all the way to the nucleus

The differential formation of the LINC-mediated perinuclear actin cap in pluripotent and somatic cells.

The actin filament cytoskeleton mediates cell motility and adhesion in somatic cells. However, whether the function and organization of the actin network are fundamentally different in pluripotent stem cells is unknown. Here we show that while conventional actin stress fibers at the basal surface of cells are present before and after onset of differentiation of mouse (mESCs) and human embryonic stem cells (hESCs), actin stress fibers of the actin cap, which wrap around the nucleus, are completely absent from undifferentiated mESCs and hESCs and their formation strongly correlates with differentiation. Similarly, the perinuclear actin cap is absent from human induced pluripotent stem cells (hiPSCs), while it is organized in the parental lung fibroblasts from which these hiPSCs are derived and in a wide range of human somatic cells, including lung, embryonic, and foreskin fibroblasts and endothelial cells. During differentiation, the formation of the actin cap follows the expression and proper localization of nuclear lamin A/C and associated linkers of nucleus and cytoskeleton (LINC) complexes at the nuclear envelope, which physically couple the actin cap to the apical surface of the nucleus. The differentiation of hESCs is accompanied by the progressive formation of a perinuclear actin cap while induced pluripotency is accompanied by the specific elimination of the actin cap, and that, through lamin A/C and LINC complexes, this actin cap is involved in progressively shaping the nucleus of hESCs undergoing differentiation. While, the localization of lamin A/C at the nuclear envelope is required for perinuclear actin cap formation, it is not sufficient to control nuclear shape

A perinuclear actin cap regulates nuclear shape

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap

The perinuclear actin cap progressively forms in hESCs following onset of differentiation.

<p>A–H. Status of apical perinuclear actin cap (A–D) and basal stress fibers (E–H) in undifferentiated hESCs at day 0 (A and E), as well as two (B and F), five (C and G), and ten days (D and G) after induction of differentiation (+VEGF and collagen IV). Red, purple, and orange arrows indicate examples of cells showing no actin cap, a disrupted/disorganized actin cap, and a well-organized actin cap, respectively. Cells were stained for differentiation marker TRA-1-81, nuclear DNA (DAPI), and actin. Scale bar, 20 µm. I. Evolution of the fraction of TRA-1-81-negative hESCs showing an actin cap. No actin cap was present in TRA-1-81-positive hESCs. J. Proportion of TRA-1-81-positive and TRA-1-81-negative hESCs showing an organized actin cap, a disorganized actin cap, or no actin cap, two and five days after onset of differentiation. K. Distribution of cells showing an actin cap in hESCs after 10 days of differentiation compared to HLFs. At least 100 cells in triplicate for a total of 300 cells were probed per condition.</p

The perinuclear actin cap in human somatic cells.

<p>A and B. Confocal microscopy sections (<i>Insets</i>) of the actin filament network at the apical surface (red), mid-height (blue), and basal surface (green) of a mouse embryonic fibroblast (MEF, panel A) and a human lung fibroblast (HLF, panel B). The main panel shows the full confocal reconstruction of the three-dimensional actin filament organization. Bottom and side panels show views along the width cross-section (bottom panel) and length cross-section (side panel) through the nucleus. Scale bar, 20 µm. C. Typical organization of the conventional basal stress fibers (left panels) and of the actin cap fibers (right panels) in a human foreskin fibroblast (HFF, top) and a human umbilical vein endothelial cell (HUVEC, bottom), as detected by epifluorescence microscopy. Scale bar, 20 µm. D. Illustrative examples of organized perinuclear actin cap, disorganized actin cap, and no actin cap in a HLF. Scale bar, 20 µm. E. Proportion of MEFs, HLFs, HFFs, and HUVECs showing an organized (orange bars), disrupted (blue bars), and no actin cap (red bars). At least 100 cells in triplicate for a total of 300 cells were probed per condition.</p

Status of Lamin A/C and LINC complexes in hESCs and iPSC during differentiation.

<p>A–D. Low- (10×, top panels) and high-magnification (60×, bottom panels) views of the organization of lamin A/C (A) and LINC complex components Nesprin2 giant (B), Nesprin3 (C), and Sun2 (D) at or near the nuclear envelope in undifferentiated hESCs and hESCs one and two days after switching to differentiation conditions. Cells were stained for nuclear DNA (DAPI), actin, TRA-1-81, and with antibodies against human lamin A/C, Nesprin2 giant, Nesprin3, and Sun2, as indicated, and visualized by immunofluorescence microscopy. Scale bar for 10× micrographs, 100 µm; Scale bar for 60× micrographs, 20 µm. E–G. Immunofluorescence micrographs showing the organization of actin and lamin A/C (E), LINC complex components Nesprin2 giant (F) and Sun2 (G) at the nuclear envelope of parental HLFs (top panels) and IPSCs (bottom panels). Scale bar, 20 µm.</p

Nuclear shaping during hESCs undergoing differentiation.

<p>A. Ensemble-averaged nuclear shape factors of undifferentiated hESCs and cells undergoing differentiation, as well as iPSCs, parental HLFs, HUVECs, and HFFs. The shape factor is close to zero for a highly elongated nucleus and unity for a perfectly round nucleus. B. Distributions of nuclear shape factors in undifferentiated hESCs and cells undergoing differentiation. At least 200 cells were probed in triplicate. C. Fraction of multi-lobulated hESCs, with a nucleus featuring at least one lobe (black curve), and showing no actin cap (red curve) as a function of days following onset of differentiation. D. Typical shapes of nuclei in undifferentiated hESCs (left panel) and cells 10 days after onset of differentiation (right panel). Scale bar, 100 µm. E. Distributions of nuclear shape factors in iPSCs, their parental HLFs, HUVECs, and HFFs. F. Fractions of multi-lobulated nuclei in undifferentiated hESCs, hESCs undergoing differentiation, iPSCs, their parental HLFs, HUVECs, and HFFs. *: P<0.05; **: P<0.01.</p

Lamin A/C is required for the proper differentiation of mouse embryonic stem cells (mESCs).

<p>A. Representative micrographs of basal (left columns) and apical (right columns) actin of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mouse embryonic stem cells at day 0 (top row), at 3 days of differentiation (middle row), and after 14 days of differentiation (bottom row), illustrating the earlier appearance of the actin cap in wildtype cells (by 3 days) when compared to heterozygotes (∼7 days) and knockouts (>14 days). All scale bars: 20 µm. B. Flow cytometry analysis of normalized stage-specific embryonic antigen 1 (SSEA1) levels of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mESCs through 14 days of differentiation. C. Replating efficiencies of <i>Lmna^+/+</i>, <i>Lmna^+/−</i>, and <i>Lmna^−/−</i> mESCs after 3, 7, and 14 days of differentiation. *: P<0.05; **: P<0.01. ***: P<0.001; ns: non-significant difference.</p