Nuclear envelope localization of human UNC84A does not require nuclear lamins

AbstractThe SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84

Substrate Adhesion Regulates Sealing Zone Architecture and Dynamics in Cultured Osteoclasts

The bone-degrading activity of osteoclasts depends on the formation of a cytoskeletal-adhesive super-structure known as the sealing zone (SZ). The SZ is a dynamic structure, consisting of a condensed array of podosomes, the elementary adhesion-mediating structures of osteoclasts, interconnected by F-actin filaments. The molecular composition and structure of the SZ were extensively investigated, yet despite its major importance for bone formation and remodelling, the mechanisms underlying its assembly and dynamics are still poorly understood. Here we determine the relations between matrix adhesiveness and the formation, stability and expansion of the SZ. By growing differentiated osteoclasts on micro-patterned glass substrates, where adhesive areas are separated by non-adhesive PLL-g-PEG barriers, we show that SZ growth and fusion strictly depend on the continuity of substrate adhesiveness, at the micrometer scale. We present a possible model for the role of mechanical forces in SZ formation and reorganization, inspired by the current data

Effect of cell shape and dimensionality on spindle orientation and mitotic timing

The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells' adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells' microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an actin dependent process. The comparison to 2D square patterns revealed that the dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells' microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells' adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis

The effect of cell shape on the cells' progression through mitosis.

<p>HeLa cells (RFP-tubulin/GFP-H2B) were synchronized and cultured on 2D homogenously coated substrates (black bars) or within square (light grey bars) or circular (dark grey bars) microwells and assessed for the time required to progress through each stage in mitosis. Cells cultured in circular microwells took longer to progress through mitosis and specifically to reach late prometaphase, than cells cultured in square microwells or on 2D substrates. All values represent time in minutes ± SEM. Key: * p<0.05, ** p<0.01; NB  =  nuclear envelope breakdown, LPM  =  late prometaphase, M =  metaphase, A =  anaphase, C =  cytokinesis.</p

The effect of the actin cytoskeleton on mitosis after culture on 2D square patterned substrates.

<p>(A) HeLa cells (YFP-paxillin) were cultured on 2D square patterns for 18 hours and imaged during interphase for actin (red) and paxillin (green). HeLa cells (RFP-tubulin/GFP-H2B) were assessed for the angle of the mitotic spindle in the xy plane at metaphase after culture on 2D square patterns and treatment with (B) media or (C) latrunculin A. A high proportion of the cells cultured on square patterns aligned the mitotic spindle along the diagonal of the cell, which was reduced by the addition of latrunculin A. (D) Conversely the addition of latrunculin A had little effect on the average orientation of the mitotic spindle perpendicular to the substrate plane. Values represent spindle angle in degrees ± SEM. (E) Furthermore, the addition of latrunculin A had little effect on the distribution of the spindle orientation. (F) Cells treated with latrunculin A (light grey bars) took significantly less time to progress from late prometaphase to metaphase and to complete mitosis than untreated cells (black bars). All values represent time in minutes ± SEM. Key: *** p<0.001; NB  =  nuclear envelope breakdown, S =  spindle formation, LPM  =  late prometaphase, M =  metaphase, A =  anaphase.</p

The role of the actin cytoskeleton in the shape dependent differences in mitosis.

<p>HeLa (YFP-paxillin) cells were cultured for 18 hours in either (A) square microwells or (B) circular microwells and imaged during interphase for actin (red) and paxillin (green); bars: 10 µm. HeLa (GFP-H2B/RFP-tubulin) cells were assessed for the parallel orientation of the mitotic spindle at metaphase after culture in (C) square or (D) circular microwells and treatment with latrunculin A for 1 hour prior to mitosis. Cells cultured in circular microwells showed a random orientation of the mitotic spindle, whilst cells cultured in square microwells predominately aligned the spindle along the long axis of the cell. (E) The inhibition of actin polymerization in HeLa (GFP-H2B/RFP-tubulin) cells lead to increased tilting in cells cultured in the square microwells. Values represent spindle angle in degrees ± SEM. (F) Perturbation of actin polymerization in cells cultured in square microwells also increased the distribution of spindle orientation, but had little effect on cells cultured in circular microwells. (G) The perturbation of the actin cytoskeleton negated the differences in mitotic timings observed between cells cultured in square microwells (light grey bars) and cells cultured in circular microwells (dark grey bars). All values represent time in minutes ± SEM. NB  =  nuclear envelope breakdown, S =  spindle formation, LPM  =  late prometaphase, M =  metaphase, A =  anaphase.</p

Analysis of mitotic timings in single cells cultured on different cell culture platforms.

<p>Analysis of mitotic timings in single cells cultured on different cell culture platforms.</p

Control of 3D cell shape using a microwell cell culture platform.

<p>(A–B) HeLa cells (RFP-tubulin/GFP-H2B) were synchronized and cultured in square (A) or circular (B) microwells for 18 hours and imaged for nucleus (green) and cell outline (Vybrant DiD cell labeling solution, red). Images show the central xy slice of the cell (top) and the corresponding xz slice of the same cell (bottom); bars: 10 µm.</p