Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape

Background

Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D) cell cultures. Cells anchored in a three-dimensional (3-D) microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D).

Methodology/Principal Findings

Arrays of 5 or 10 µm deep microwells were fabricated in polydimethylsiloxane (PDMS). The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D) or trapped in microwells (3-D) of controlled size, shape, and wall rigidity. On rigid substrates (Young''s Modulus = 1 MPa), cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area) and total surface areas of adhesion (microwell bottom plus wall surface area) that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa), regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant.

Conclusion/Significance

These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.     

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.     

The novel actin/focal adhesion-associated protein MISP is involved in mitotic spindle positioning in human cells

Accurate mitotic spindle positioning is essential for the regulation of cell fate choices, cell size and cell position within tissues. The most prominent model of spindle positioning involves a cortical pulling mechanism, where the minus end-directed microtubule motor protein dynein is attached to the cell cortex and exerts pulling forces on the plus ends of astral microtubules that reach the cortex. In nonpolarized cultured cells integrin-dependent, retraction fiber-mediated cell adhesion is involved in spindle orientation. Proteins serving as intermediaries between cortical actin or retraction fibers and astral microtubules remain largely unknown. In a recent genome-wide RNAi screen we identified a previously uncharacterized protein, MISP (C19ORF21) as being involved in centrosome clustering, a process leading to the clustering of supernumerary centrosomes in cancer cells into a bipolar mitotic spindle array by microtubule tension. Here, we show that MISP is associated with the actin cytoskeleton and focal adhesions and is expressed only in adherent cell types. During mitosis MISP is phosphorylated by Cdk1 and localizes to retraction fibers. MISP interacts with the +TIP EB1 and p150^glued, a subunit of the dynein/dynactin complex. Depletion of MISP causes mitotic arrest with reduced tension across sister kinetochores, chromosome misalignment and spindle multipolarity in cancer cells with supernumerary centrosomes. Analysis of spindle orientation revealed that MISP depletion causes randomization of mitotic spindle positioning relative to cell axes and cell center. Together, we propose that MISP links microtubules to the actin cytoskeleton and focal adhesions in order to properly position the mitotic spindle.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain

The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.     

SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation

Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation.     

Integrin-mediated adhesion orients the spindle parallel to the substratum in an EB1- and myosin X-dependent manner

The orientation of mitotic spindles is tightly regulated in polarized cells, but it has been unclear whether there is a mechanism regulating spindle orientation in nonpolarized cells. Here we show that integrin-dependent cell adhesion to the substrate orients the mitotic spindle of nonpolarized cultured cells parallel to the substrate plane. The spindle is properly oriented in cells plated on fibronectin or collagen, but misoriented in cells on poly-L-lysine or treated with the RGD peptide or anti-beta1-integrin antibody, indicating requirement of integrin-mediated cell adhesion for this mechanism. Remarkably, this mechanism is independent of gravitation or cell-cell adhesion, but requires actin cytoskeleton and astral microtubules. Furthermore, myosin X and the microtubule plus-end-tracking protein EB1 are shown to play a role in this mechanism through remodeling of actin cytoskeleton and stabilization of astral microtubules, respectively. Our results thus uncover the existence of a mechanism that orients the spindle parallel to the cell-substrate adhesion plane, and identify crucial factors involved in this novel mechanism.     

Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

F-actin in mitotic spindles of synchronized suspension culture cells of tobacco visualized by confocal laser scanning microscopy

TRITC-labelled phalloidin was used to visualize F-actin distribution during mitosis in Nicotiana tabacum BY-2 suspension cells. Aphidicolin was used to synchronize cell suspensions, which enabled sufficient numbers of mitotic cells to be obtained. F-actin was present in the spindle, and its orientation seemed to correlate with the known microtubular arrays. The use of confocal microscopy greatly reduced background fluorescence, and therefore fine actin filaments could be observed in spindles previously thought to be devoid of actin.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

The cilia protein IFT88 is required for spindle orientation in mitosis

Cilia dysfunction has long been associated with cyst formation and ciliopathies. More recently, misoriented cell division has been observed in cystic kidneys, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells. Several IFT proteins also localize to spindle poles in mitosis, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737(orpk) and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.     

Aurora B‐dependent polarization of the cortical actomyosin network during mitotic exit

The isotropic metaphase actin cortex progressively polarizes as the anaphase spindle elongates during mitotic exit. This involves the loss of actomyosin cortex from opposing cell poles and the accumulation of an actomyosin belt at the cell centre. Although these spatially distinct cortical remodelling events are coordinated in time, here we show that they are independent of each other. Thus, actomyosin is lost from opposing poles in anaphase cells that lack an actomyosin ring owing to centralspindlin depletion. In examining potential regulators of this process, we identify a role for Aurora B kinase in actin clearance at cell poles. Upon combining Aurora B inhibition with centralspindlin depletion, cells exiting mitosis fail to change shape and remain completely spherical. Additionally, we demonstrate a requirement for Aurora B in the clearance of cortical actin close to anaphase chromatin in cells exiting mitosis with a bipolar spindle and in monopolar cells forced to divide while flat. Altogether, these data suggest a novel role for Aurora B activity in facilitating DNA‐mediated polar relaxation at anaphase, polarization of the actomyosin cortex, and cell division.     

Surface and shape changes during cell division

Summary Rat kangaroo cells (PtK₂) were studied with scanning and transmission electron microscopy in order to correlate shape changes during the cell cycle with the presence or absence of microvilli and stress fibers. During interphase, bundles of actin are prominent in the cytoplasm, and microvilli are localized over and around the centrally positioned nucleus. As mitosis begins, the interphase bundles of actin and the microvilli disappear, but the mitotic cells maintain a flattened shape. At metaphase the cell is still so flat that both the chromosomes and spindle apparatus are visible through the intact cell membrane. Microvilli reappear in late anaphase above the chromosomes and poles. Before cleavage begins, microvilli increase in number until they cover the apical surface of the cell. At the same time, the cell increases in height so that the chromosomes and mitotic apparatus can no longer be detected through the cell membrane. During cleavage, microvilli continue to cover the cell in a uniform manner but become greatly diminished in number after cytokinesis is completed and the cells flatten and enter interphase. It is suggested that the microvilli organize a network of actin filaments which interact with cortical myosin to produce the cell rounding prior to cleavage.     

Dlg1 controls planar spindle orientation in the neuroepithelium through direct interaction with LGN

Oriented cell divisions are necessary for the development of epithelial structures. Mitotic spindle orientation requires the precise localization of force generators at the cell cortex via the evolutionarily conserved LGN complex. However, polarity cues acting upstream of this complex in vivo in the vertebrate epithelia remain unknown. In this paper, we show that Dlg1 is localized at the basolateral cell cortex during mitosis and is necessary for planar spindle orientation in the chick neuroepithelium. Live imaging revealed that Dlg1 is required for directed spindle movements during metaphase. Mechanistically, we show that direct interaction between Dlg1 and LGN promotes cortical localization of the LGN complex. Furthermore, in human cells dividing on adhesive micropatterns, homogenously localized Dlg1 recruited LGN to the mitotic cortex and was also necessary for proper spindle orientation. We propose that Dlg1 acts primarily to recruit LGN to the cortex and that Dlg1 localization may additionally provide instructive cues for spindle orientation.     

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.     

NuMA localization,stability, and function in spindle orientation involve 4.1 and Cdk1 interactions

The epidermis is a multilayered epithelium that requires asymmetric divisions for stratification. A conserved cortical protein complex, including LGN, nuclear mitotic apparatus (NuMA), and dynein/dynactin, plays a key role in establishing proper spindle orientation during asymmetric divisions. The requirements for the cortical recruitment of these proteins, however, remain unclear. In this work, we show that NuMA is required to recruit dynactin to the cell cortex of keratinocytes. NuMA''s cortical recruitment requires LGN; however, LGN interactions are not sufficient for this localization. Using fluorescence recovery after photobleaching, we find that the 4.1-binding domain of NuMA is important for stabilizing its interaction with the cell cortex. This is functionally important, as loss of 4.1/NuMA interaction results in spindle orientation defects, using two distinct assays. Furthermore, we observe an increase in cortical NuMA localization as cells enter anaphase. Inhibition of Cdk1 or mutation of a single residue in NuMA mimics this effect. NuMA''s anaphase localization is independent of LGN and 4.1 interactions, revealing two distinct mechanisms responsible for NuMA cortical recruitment at different stages of mitosis. This work highlights the complexity of NuMA localization and reveals the importance of NuMA cortical stability for productive force generation during spindle orientation.     

Doublecortin induces mitotic microtubule catastrophe and inhibits glioma cell invasion

Doublecortin (DCX) is a microtubule (MT) binding protein that induces growth arrest at the G2–M phase of cell cycle in glioma and suppresses tumor xenograft in immunocompromised hosts. DCX expression was found in neuronal cells, but lacking in glioma cells. We tested the hypothesis that DCX inhibits glioma U87 cell mitosis and invasion. Our data showed that DCX synthesizing U87 cells underwent mitotic MT spindle catastrophe in a neurabin II dependent pathway. Synthesis of both DCX and neurabin II were required to induce apoptosis in U87 and human embryonic kidney 293T cells. In DCX expressing U87 cells, association of phosphorylated DCX with protein phosphatase-1 (PP1) in the cytosol disrupted the interaction between kinesin-13 and PP1 in the nucleus and yielded spontaneously active kinesin-13. The activated kinesin-13 caused mitotic MT catastrophe in spindle checkpoint. Phosphorylated-DCX induced depolymerization of actin filaments in U87 cells, down-regulated matrix metalloproteinases-2 and -9, and inhibited glioma U87 cell invasion in a neurabin II dependent pathway. Thus, localization of the DCX–neurabin II–PP1 complex in the cytosol of U87 tumor cells inhibited PP1 phosphatase activities leading to anti-glioma effects via (1) mitotic MT spindle catastrophe that blocks mitosis and (2) depolymerization of actin that inhibits glioma cell invasion.     

Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells

Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. In this study, we show that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase-dependent phosphorylation. Activated moesin homogenously localizes at the cortex in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. We propose that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division.     

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.