SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells

The HOF structures of nitrotetraphenylethene derivatives provide new insights into the nature of AIE and a way to design mechanoluminescent materials

This study probes the effect of intramolecular rotations on aggregation-induced emission (AIE) and leads to a kind of supramolecular mechanoluminescent material. Two hydrogen-bonded organic frameworks (HOFs), namely HOFTPE3N and HOFTPE4N, have been constructed from nitro-substituted tetraphenylethene (TPE) building blocks, namely tris(4-nitrophenyl)phenylethene (TPE3N) and tetrakis(4-nitrophenyl)ethene (TPE4N). Using single-crystal X-ray diffraction analysis, two types of pores are observed in the HOFTPE4N supramolecular structure. The pore sizes are 5.855 Å × 5.855 Å (α pores) and 7.218 Å × 7.218 Å (β pores). Powder X-ray diffraction and differential scanning calorimetry studies further reveal that the α pores, which contain nitrophenyl rings, quench the emission of HOFTPE4N. This emission can be turned on by breaking the α pores in the HOFs by grinding the sample. Temperature-dependent emission studies demonstrate that the emission quenching of HOFTPE4N is attributed to the intramolecular rotations of nitro-substituted phenyl units within the space of the α pores. These results clearly reveal AIE by controlling the intramolecular rotations, which can serve as a basis for developing mechanoluminescent materials

A Sensitized Screen for Genes Promoting Invadopodia Function In Vivo: CDC-42 and Rab GDI-1 Direct Distinct Aspects of Invadopodia Formation.

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue

SPARC functions extracellularly to reduce BM type IV collagen levels.

<p>(A) Neuronally expressed SPARC (<i>rab-3>SPARC</i>::<i>GFP</i>; top) is found at the BM (collagen::mCherry, center; merge, bottom). (B) Graph depicts the frequency of ACs breaching the BM in <i>unc-40(e271)</i> and vulvaless (<i>lin-3(n1059)/lin-3(n378)</i>) animals when SPARC is overexpressed in the neurons (<i>rab-3>SPARC</i>::<i>GFP</i>) compared to worms not overexpressing SPARC (n≥49 for each genotype). *** denotes p<0.0005 by two-tailed Fisher’s exact test, error bars show 95% confidence intervals with a continuity correction. (C) A spectral representation of collagen::mCherry fluorescence at the BM when SPARC is expressed neuronally is shown on the left and the quantification of collagen fluorescence at the BM is shown on the right (n = 15 animals for each treatment). ** denotes p<0.005 by two-tailed unpaired Student’s <i>t</i>-test; error bars show SEM. Scale bars denote 5 μm.</p

SPARC overexpression promotes AC invasion by decreasing type IV collagen levels in the BM.

<p>(A) A spectral representation of the fluorescence intensity shows collagen::mCherry (left) and laminin::mCherry (right) fluorescence at the BM in wild type (top) and SPARC overexpression (o/e; <i>syIs115;</i> bottom) animals. The graph below depicts the average levels of fluorescence for collagen::mCherry and laminin::mCherry at the BM (n≥16 for each treatment). (B) Animals expressing no excess SPARC, <i>hsp>SPARC</i> or <i>hsp>SPARC</i>^{<i>R152L</i>,<i>Q159A</i>} were subjected to a two hour heat shock to drive high levels of SPARC expression. The graph shows average collagen fluorescence at the BM before heat shock and after 4 hours of recovery (6 hours after the start of SPARC induction; n≥21 for each treatment). (C) The graph depicts the frequency of BM breach in animals treated with RNAi targeting the <i>emb-9</i> collagen α1 subunit (n≥50 for each treatment). Error bars denote the standard error of the mean (SEM, A and B) or 95% confidence intervals with a continuity correction (C). *** denotes p<0.0005 by unpaired two-tailed Student’s <i>t</i>-test (A and B) or two-tailed Fisher’s exact test (C). Scale bars denote 5 μm.</p

SPARC regulates the transport of type IV collagen during development.

<p>(A) DIC images and corresponding spectral representations of fluorescence intensity showing collagen::mCherry in the pharyngeal BM of a wild type animal (top panel) and SPARC RNAi-treated animal (bottom). Arrowheads indicate BM deformations after SPARC reduction. The average collagen::mCherry and laminin::GFP fluorescence levels at the pharyngeal BM in wild type and SPARC RNAi-treated animals are quantified in the graph on the right and compared using a two-tailed unpaired Student’s <i>t</i>-test (n≥15 for each treatment; *** denotes p<0.0005 for collagen::mCherry; p = 0.3 for laminin::GFP; error bars show SEM). (B) Representative fluorescence images of collagen::mCherry in the Z-plane of body wall muscle cells in a wild type animal (top, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005905#pgen.1005905.g002" target="_blank">Fig 2</a> for the location of body wall muscles in cross section) and SPARC RNAi-treated animal (bottom). The boxed regions indicate body wall muscle cells and are magnified on the right with dashed lines representing the muscle surface as determined by corresponding DIC images. Collagen::mCherry accumulated abnormally at the muscle surface in the SPARC RNAi-treated animal (arrowheads), and was difficult to detect at the muscle surface in the wild type animal. The average collagen::mCherry fluorescence levels at the muscle surface (dashed yellow lines on left) are quantified in the graph on the right (n = 6 wild type and 12 RNAi treated animals). *** denotes p<0.0005 by two-tailed unpaired Student’s <i>t</i>-test; error bars show SEM. (C) Spectral representation of collagen::mCherry fluorescence intensity at the gonadal BM in wild type (top) and SPARC RNAi treated (bottom) representative animals. The average collagen::mCherry fluorescence levels are quantified in the graph on the right (n≥40 for each treatment; p = 0.8 by unpaired two-tailed Student’s <i>t</i>-test; error bars show SEM). (D) Graph depicts the frequency of ACs breaching the BM in <i>unc-40(e271)</i> animals treated with SPARC RNAi (n≥51 for each treatment; p = 1.0 by two-tailed Fisher’s exact test). Error bars show 95% confidence intervals with a continuity correction. Scale bars denote 5 μm.</p

SPARC overexpression slows type IV collagen recovery in the BM.

<p>Half of the uterine tissue was photobleached by exposing collagen::mCherry within this region to 561 nm light for two minutes (0 min recovery, left; arrow denotes region of bleach, arrowhead denotes unbleached control region). Collagen::mCherry fluorescence recovery was assessed two hours later (120 min, right). Average collagen::mCherry fluorescence levels for the control and bleached regions at 0 min and 120 min is graphed below (error bars denote SEM). An average of 44±4% of the total collagen recovered in wild type and 25±3% of collagen in worms overexpressing SPARC (n≥10 for each condition, p<0.005 by two-tailed unpaired Student’s <i>t</i>-test). Scale bar denotes 5 μm.</p