αII-Spectrin is present in the invadosomes of different cell systems Previous work localized αII-spectrin in podosome-like structures of non-invasive carcinoma cells. To expand this observation, we determined the localization of endogenous αII-spectrin in different invadosome models such as in human endothelial cells (HMEC-1 cell line), primary human endothelial cells (HUVEC) after different treatments (PMA or EGF) leading to invado- some formation, and in mouse embryonic fibroblasts expressing a constitutively activated mu- tant of the non-receptor tyrosine kinase Src (SrcY527F). HMEC-1 cells were treated for one hour with PMA (50 ng/ml), a well-known agent that induces invadosome assembly via PKC activation, and approximately 15% of cells were able to assemble invadosome structure-like rings as revealed by cortactin staining (red, Fig. 1A). In non-treated HMEC-1, αII-spectrin staining (green, Fig. 1A) was mostly localized in the cytoplasm and in the membrane at the edge of some cell protrusions. PMA treatment induced the recruitment of αII-spectrin in inva- dosome rings and localized nicely with cortactin. In a similar way, αII-spectrin relocalized into invadosomes of HMEC-1 cells that were induced by either EGF (5 ng/ml, S1A Fig.) or TGFβ (5 ng/ml, S1B Fig.). The presence of αII-spectrin in invadosomes seems to be a general feature as αII-spectrin was also found in invadosomes of MEF cells expressing the constitutively active mutant of Src, SrcY527F (Fig. 1A). Although invadosome cores are difficult to localize in these models, αII-spectrin did not co-localize with cortactin hot spots that are present in invadosome rings. Instead, αII-spectrin appeared to localize to the actin cloud surrounding the actin core (zoom Fig. 1A and B). As invadosomes are dome-shaped structures extending orthogonally from the basement membrane, αII-spectrin was localized in3D reconstructed invadosome. Based on orthogonal projections of invadosome rings in SrcY527F-MEFs, αII-spectrin was mostly present at the basis of the invadosomes. This confirms αII-spectrin localization in the actin cloud rather than in the core, and suggests a potential function for this protein at the in- terface between the membrane and actin cytoskeleton of the invadosome.
Different antibodies can bind different epitopes of the same protein, or even distinguish between native and denatured proteins. We separated puriied human proteins and bovine disc extracts by SDS-PAGE and performed a Western blot using culture media of human degenerated discs as the primary polyclonal antibody probe. The assay conirmed the presence of IgGs principally against the monomeric form of collagen type II and similar, but weaker signal, against collagen type I. Involvement of collagen type II in the autoimmune reaction is highly probable; IVD degeneration has been correlated with increased degradationof collagen II (Antoniou et al., 1996), yet collagen II might trigger articular cartilage diseases (Burkhardt et al., 2002) and antibodies to denatured collagens have been found in patients with rheumatoid arthritis (RA) (Morgan et al., 1987). We also found antibodies against the minor ibrillar human collagen type V, which plays a role in the organisation of heterotypic type I/V collagen ibrils (Birk, 2001). Interestingly strong signal against native collagen type V was observed only with dot blot assay, but not with SDS electrophoresis and Western blot, probably because under denaturing conditions collagen V epitopes are no longer properly recognised by speciic antibodies. Identifying antibodies against all these proteins raises the question if autoimmune reactivity in the disc is not generic to the whole collagen family? Probably not, as we could not ind IgGs against collagen type III, a collagen present in outer AF of degenerated IVDs and correlated with altered metabolism of disc tissue (Adam and Deyl, 1984).
In agreement with others, our data demonstrate that TM cells are capable of ingesting a vast variety of materials for an extended period of time without compromising cell viability [5–11]. Although we did not observe a marked preference between phagocytic substrates (opsonized versus non- opsonized, biotic versus nonbiotic), the maturation of phagosomes appears to differ depending on the contained particle. Thus, whereas E.coli-containing phagosomes showed 100% co-localization with the lysosomal marker LTR, thus indicating the maturation into phagolysosomes, inert latex beads did not. We cannot discount that the presence of non- biotic material might affect the uptake of the tracer or the phagolysosomal pH; however, electron micrographs showed that both latex beads and pigment particles preferentially existed within the cells in isolated phagosomes. Interestingly, collagen I-coated beads could be found in different intermediates maturation steps: as isolated phagosomes, non- stained by LTR; as isolated phagolysosomes, displaying LTR fluorescence surrounding the surface membrane; and by electron microscopy, as mature merged autophagolysosomes. Maturation of phagolysosomes seemed to be associated with higher lysosomal content as quantified by LTR and LAMP1 content. Similar results have been reported in macrophages when comparing the maturation of phagosomes containing opsonized sheep erythrocytes, biodegradable poly-e- caprolactone microspheres, and non-biodegradable polystyrene microspheres .
The proteins coded by MMP1 and MMP3 belong to the matrix metalloproteinases (MMPs) family, a group of zinc-containing endopeptidases that actively participate in the degradationof components of the extracellularmatrix (ECM). MMPs have different but overlapping substrate specificity. In particular, MMP1 preferentially cleaves collagen I, whereas MMP3 shows higher specificity for collagen IV [23,24]. To evaluate whether the observed transcriptional up-regulation of MMP1 and MMP3 might translate into increased collagenolytic activity upon phagocytosis, we monitored the degradationof collagen I and collagen IV in phagocytically challenged porcine TM cells using the quenched fluorescent substrates DQ-collagen I and DQ- collagen IV, respectively. As seen in Figure 7A, phagocytosis of E. coli promoted the degradationof collagen I overtime, demon- strated by the increased in fluorescence resulting from the proteolytic degradationof the substrate. The presence of pigment particles, however, caused a slightly decrease in collagenolytic I activity compared to control. No significant changes in the degradationof collagen IV were observed with any of the phagocytic ligands at the time tested (Figure 7B).
The aim of this project is to develop a new hydrogel made of Dextrin (Dex), Hyaluronic acid (HA) and Extracellularmatrix (ECM) from Small Intestine Submucosa (SIS), using different types of HA, mixed different proportions. In this study, HA and Dex were oxidized by sodium periodate to create aldehyde functional groups, which could be cross-linked by Adipic Acid Dihidrazide (ADH). Their characterization was performed based on gelation period and degradation rate. In addition, cell viability tests were performed through a Resazurin assay, a MTS assay and Live and Dead (LD) using osteoblastic cell line MC3T3 calvaria from mouse. Results showed a low gelation time for all the hydrogels and low degradation rates for mixed hydrogels with high contents of high molecular weight (MW) HA. The degradation tests demonstrated that the selected hydrogel could maintain the gel matrix over 70 days.
Accumulation of connective tissue, particularly extracellularmatrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and−3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFβ and CTGF; MMP-2 and-9; TIMP-1 and−2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradationof the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.
Since lung cancer is the phenotypic consequence of the interactions between epithelial and mesenchymal factors (such as extracellularmatrix, ECM) for growth, invasion, and metastasis, ongoing studies are currently validating other biomarkers. In this context, remarkable qualitative and quantitative modifications of the epithe- lium-mesenchymal components, such as hyaluronan (HA), E-cadherin adhesion molecules, and the transform- ing growth factor b (TGF-b) may favor invasion, cellular motility, and proliferation. HA, a large unsulfated glycos- aminoglycan, is expressed by many cell types and contributes to cellular functions. The biosynthesis and turnover of HA is abnormal in many types of diseases and inflammatory processes. Elevated levels of HA in serum and tissues are an important marker for a variety of pathological conditions (6). Migration of tumor cells in a cellular matrix rich in HA occurs after the matrix is degraded into fragments of 2-25 disaccharides and is mediated by specific cell surface receptors, CD44, and receptor for HA-mediated motility (7). Hyaluronan synthase (HAS) consists of a group of isozymes responsible for the synthesis of HA with different chains. There are 3 types of HAS termed HAS-1, HAS-2, and HAS-3 (8). HAS mRNA levels often correspond to the rate of HA synthesis, and are known to influence the content of HA in transplanted tumors (9). Therefore, upregulation of HAS expression is a likely contributor to HA accumulation in tissues, and promotes tumor growth (10) and metas- tasis in experimental animals, especially when coex- pressed with Hyal (11). Different types of hyaluronidases (HAases), Hyal-1, Hyal-2, and Hyal-3, can degrade the long chains of alternating units of N-acetylglucosamine and glucuronic acid into different sizes. For example, Hyal-1 is responsible for degrading HA into small fragments of 20-25 disaccharides needed to activate the MAP-kinase pathway (12). The major transcript of Hyal-3 is enzymatically inactive and appears to have only a supportive role in Hyal-1 expression (13). Increased HAase expression has also been reported in colon (14) and prostate cancer (15), as well as in breast tumor metastases (16). Notably, the growth of murine lung carcinoma and melanoma cells is influenced by Hyal-1 (17). In lung fibroblasts, HA synthesis and degradation appear to be controlled by TGF-b1 (18). TGF-b has profound growth-suppressive effects on normal epithelial cells, but supports metastasis formation in many tumor types. As opposed to the function of HAases, TGF-b is a potent stimulator of ECM protein (19). The key molecule that maintains epithelial cell-cell adhesion and integrity is E-cadherin. E-cadherin, the prototype member of the classical cadherin family, is a Ca 2+ -dependent transmem- brane receptor that mediates cell-cell adhesion at the
Matrix metalloproteinases (MMP) are extracellular proteolytic enzymes (endopep- tidases) capable of digesting various struc- tural components of the ECM. Growth, in- vasion and metastatic potential of solid tu- mors depend on the formation and develop- ment of new blood vessels concomitant with the degradationof the ECM. It was shown that ALA and light induced MMP-1 and MMP-3 expression in normal and sclero- derma fibroblasts, besides reducing collagen type I mRNA expression (53). Also, after repeated PDT, lesional skin biopsies of pa- tients with localized scleroderma showed a marked induction of MMP-1 in the dermis (54); a paracrine mechanism seemed to oc- cur, as evidenced by the induction of MMP- 1 and MMP-3 protein levels of fibroblasts stimulated with previously PDT-treated ke- ratinocyte medium. Thus, induction of col- lagen-degrading enzymes together with a reduction of collagen production was thought to be responsible for the antisclerotic effects of ALA-PDT observed in vivo (55). PDT can also increase both the expression and the enzymatic activity of MMP-9 in BA tumors (56). The inflammatory response and host cell infiltration, described previously, sug- gest that the increased expression of MMP-9 observed in tumor tissue after PDT involves the influx of MMP-9-expressing inflamma- tory host cells, as opposed to direct PDT- induced expression of MMP-9 in inflamma- tory cells present within tumor tissue at the time of treatment. These results demonstrate that EC and infiltrating host cells are sources of MMP-9 in PDT-treated BA tumors (56).
Besides the knowledge of the effect of IL-1 on cellular gene expression and the understanding of the molecular mechanisms of IL-1, signal transduction in chondrocytes – including possible crosstalks – is increasingly important. In particular, this can expose targets specifically to modify adverse effects of IL-1ß without affecting the whole spectrum of physiological function of this molecule within intact cartilage, which might be functionally important – as discussed below. These molecular mechanisms of IL-1 action have been studied extensively in other cell systems. IL-1 acts through the three classical MAPK-signalling pathways, namely ERK (extracellular signal regulated kinase), p38, and JNK (Jun terminal kinase), as well as NFkB (nuclear factor of kappa light polypeptide gene enhancer in B-cells) (for review see Saklatvala, 2006) (fig. 3). Of interest, whereas IL-1ß can activate all four pathways, the regulation of specific genes involves only a subset of these and this to a variable extent. Thus, the ERK- pathway appears to be particularly important for the induction of other cytokines such as IL-6 and LIF (Fan et al., 2004a), whereas ERK and NFkB together regulate anabolic and catabolic genes such as collagen type II and MMP-1 and -13 (Barchowsky et al., 2000; Mengshol et al., 2000; Fan et al., 2006). The latter appears also to implicate signalling through p38.
Apart from adverse effects on mechanical stability and electrical conduction, proteolytic fragments liberated from BM constituents are biologically active by inhibiting angiogenesis. The early presence of these fragments following mild hypothermic I/R may potentially suppress vascularization and formation of new vessels in the re-perfused heart, thus contributing to the development of post-ischemic heart failure. For example, endo- statin, the 25 kDa-fragment of collagen XVIII, has been characterized in experimental studies and its pro- and anti- angiogenic effects have been reported [21,26,34]. Several other matrix-derived degradation products have recently been reported and mediate modulating function in post-ischemic remodelling of the heart. TUM, ARR and LG3 are known as potent inhibitors of angiogenesis. Due to their modulating function, the dynamic changes of release of these fragments are of particular interest during early reperfusion after myocardial ischemia, as this period may offer opportunities for additional therapy in the clinical setting. We recently characterized the in vivo post-ischemic release of endostatin by collagen XVIII degradation during early ischemia-reperfusion and demonstrated the highly dynamic changes of release ofmatrix cleavage in this time period . Endostatin is currently undergoing preclinical trials to investigate its therapeutic potential in tumor therapy [1,18].
Much remains to be determined about the nature of VEGF proteolysis and cell-mediated release. For example, endothelial cells [3,19], neutrophils , and macrophages [17,30–32] all have been implicated as potential mediators of VEGF release, but when each cell type is important is not known. Thus the question of when VEGF release is an autocrine or paracrine process has not been answered. In addition, the extent of VEGF release is system- dependent. Lee et al. reported that in the serum of mice implanted with fibrosarcoma, over 80% of circulating VEGF is in a cleaved form , a value similar to that in retinal tissue of mice with oxygen-induced retinopathy . A lower, but still significant value of ,30% cleaved VEGF was detected in human ovarian tumor lysates . In contrast, in an in vitro fibrin-based system, a significant release-dependent cellular response occurred without any detectable VEGF release . We do not currently know the rate at which VEGF release or cleavage occurs in biology. Experiments treating growth factors with exogenous protease show that 20 min with 400 nM plasmin [2,6,34], 30 min with 20 m M elastase , or 24 h with ,0.3 to 20 nM active MMP9 [1,8] all carry significant proteolytic potential. However, in vivo protease levels in plasma and in pathological fluid samples are typically lower, between 100 pM–20 nM [36–41], while clearance rates for VEGF (after accounting for its proteolytic degradation) are very rapid, ,1 h . It is not currently known if these protease levels are sufficient to account for the .80% circulating cleaved VEGF .
Collagen fibers constitute the main component of the ECM and are a fibrous protein that consists of three α- chains, which form a rope-like triple helix, providing ten- sile strength to the ECM (Montes 1996). Despite their broad diversity in the connective tissue, types I, II, III (fibrillar) and IV, V, VI (non fibrillar or amorphous) rep- resent the main collagen fibers. Type III collagen fiber is more flexible and susceptible to breakdown, type I collagen is comprised of thicker more cross-linked fib- ril (Rocco et al. 2001, Santos et al. 2006), whereas type IV collagen fiber is located mainly in basement mem- brane. Their turnover is a dynamic process, necessary to the maintenance of the normal lung architecture (Rocco et al. 2003). The final collagen accumulation does not depend only on its synthesis, but also, on its degradation (Rocco et al. 2001, 2003). Consequently, the ECM is a dynamic structure, and equilibrium between synthesis and degradationof ECM components is required for the maintenance of its homeostasis (Santos et al. 2006).
We achieved human liver ECM scaffold isolation by establishing a two-step protocol that requires a liver biopsy immersed in differential buffers and then subjected to gentle and con- tinuous agitation (Fig 1A). In the first step, the blood was flushed out from the liver biopsy by means of a washing buffer, thus turning the color of the tissue to yellowish-brown in 24 hours (Fig 1B, upper panels). In the second step, after 48 hours of decellularization buffer, a translu- cent acellular scaffold, which retained the gross shape of a liver biopsy, was generated (Fig 1B, upper panels). Notably, similar results were obtained applying this protocol to mouse liver lobes, thus indicating that the effectiveness of the method is not limited to the biopsy speci- mens size (Fig 1B, lower panels). Western blotting analysis provided evidence of the successful isolation of liver bioscaffold highly enriched for ECM components (Fig 1C). Mass spectrome- try analysis highlighted that collagens represented more than 60% of the human liver ECM scaffold molecules, while non-collagenous proteins and proteoglycans contributed to 28,81% and 6,35%, respectively. (Fig 1D and S1 Table). As expected, the analysis of total collagen components showed a predominant abundance of fibrillar collagen type I (COL1A1 and COL1A2) (S1 Fig). Interestingly, five of the collagen proteins (COL2A1, COL21A1, COL23A1, COL5A3 and COL26A1) were found in the matrixof human adult liver. To the best of our knowledge, this study represents the first evidence of these proteins in the matrixof human adult liver, thus further highlighting the efficacy of the ECM scaffold isolation procedure.
These findings have clinical relevance in the pathogenesis of dental caries and development of novel antibiofilm approaches. The shear rate in the oral cavity due to salivary flow is rel- atively low (Bourne, 2002), which could explain why EPS- mediated biofilm build-up persists on tooth surfaces and is difficult to detach under salivary flow. Thus, novel devices to enhance mechanical removal of biofilms can be developed based on knowledge about the biophysical properties of the biofilm. Recently, a prototype AirFloss instrument that generates high shear stress locally was capable of removing sucrose-grown S. mutans biofilms from the interproximal space (Rmaile et al., 2014). Furthermore, enzymes or compounds capable of alter- ing the viscoelastic properties of the biofilm could be effec- tive to prevent biofilm-dependent diseases (Daniels et al., 2010; Kostakioti et al., 2013; Nguyen et al., 2014). The possibility of using glucanohydrolases as therapeutic approach against dental caries has been explored (e.g., Bowen, 1972; Guggenheim et al., 1980) despite limitations in the clinical setting, possibly due to
Importantly, Shh has been found to depend on matrix components, such as proteoglycans and vitronectin, in order to be presented to target cells in the developing mouse brain (Pons and Marti, 2000; Chan et al., 2009). A similar situation might happen in the context of the developing somite, with the fibronectin binding to the Shh morphogen and presenting the signaling molecule to target cells. On the other hand, affecting the fibronectin matrix might also affect other extracellularmatrix components that could be responsible for presenting Shh in the target cells. One possible candidate could be laminin, as referred previously. Laminins and their receptors, particularly α6β1, are known to interact with Shh in the developing cerebellum, and this interaction is important for the correct patterning of granule precursor cells. α6β1 integrin clusters the Shh bound to laminin in the target cell membrane, close to Patched-Smoothened complexes (Blaess et al., 2004). Therefore, normal ECM-integrin ligation and signaling in the paraxial mesoderm might be important for the correct clustering of Shh receptors in the cell membrane of somite cells, leading to a correct interpretation of the signal that is being produced normally by the axial structures.
Fig 1. ECM stiffness regulates the cellular phenotype of human mesenchymal stem cells (hMSCs) and the localization and transcriptional activity of TAZ. (A) In indicated hydrogels, cell adhesion and morphology were visualized by light microscopy. Bright field images were taken 24hr after seeding. (B) ECM stiffness controls focal adhesion complex formation and TAZ localization. hMSCs were immunostained with an anti-vinculin antibody to detect focal adhesions (green fluorescence signal) 24 h after seeding. TAZ localization was visualized as a red fluorescence signal. DAPI was used to stain the cell nucleus. (C) The expression of TAZ target genes, including CTGF and CYR61, were analyzed by qRT-PCR using the cells in panel (A). Target gene expression was normalized to the GAPDH expression. Data is shown as fold induction. Asterisks indicate statistical significance (***p < 0.005, t-test). (D) The luciferase reporter gene CTGF-luc was introduced into hMSCs. After 16 h, the transfected cells were plated on 1.37 or 4.47 kPa hydrogels. After 24 h, luciferase reporter gene activity was analyzed. The pGL3-basic luciferase reporter gene, which has no promoter for transcription, was used as a negative control. A Renilla luciferase-expressing vector was used as a transfection control. Luciferase activity was normalized to Renilla luciferase activity and is expressed as relative fold induction. (***p < 0.005, t-test). (E) hMSCs were seeded on a 1.37 or 4.47 kPa hydrogel, and twenty four hours after seeding, total RNAs were isolated and qRT-PCR analysis was assessed to see the expression of TAZ gene. Gene expression was normalized to GAPDH.
compared to the paired normal ECM. This trend was also observed in HCT-15 cells but to a less extent. Overall, the increase in CD133 expression, both concerning the number of positive cells and number of molecules per cell, is the most consistent and points to a shift in cancer cell stem-like profile under the influence of tumor ECM. Recently, CD133 expression was associated with poor prognosis in stage II colorectal carcinoma, in a cohort of more than 300 patients who had curative surgical resection (162) and, in another study, knockout of CD133 in colon cancer cells by CRISPR/Cas9 gene editing resulted in significant reduction of EMT and colony formation (163). Nevertheless, the combination of at least two cell surface markers usually provides a more reliable CSC identification and, in fact, our results show a significant increase in all double positive populations, upon tumor ECM instruction. CD44 is a particularly important molecule in what concerns cell-matrix interactions since its main ligand is hyaluronic acid (HA), an abundant component of the ECM (164), that upon CD44 activation results in the activation of signaling pathways involved in proliferation, cell survival, cytoskeletal alterations and motility. Additionally, colorectal tumours have increased HA levels when compared to normal tissues (165), and we know that the decellularization protocol used in this study was previously shown not to affect, qualitatively, HA is certainly present in the tissue samples, as we and others observed (28).
Background: Epithelial–mesenchymal transition (EMT) is defined as a transformation of tubular epithelial cells into mesenchymal ones. These cells migrate through the extracellularmatrix and change into active myofibroblasts, which are responsible for excessive matrix deposition. Such changes may lead to tubular dysfunction and fibrosis of the renal parenchyma, characteristic of chronic kidney disease (CKD). However, there are no data on potential EMT markers in children with CKD. The aim of our study was to assess the usefulness of fractional excretion (FE) of survivin, E-cadherin, extracellularmatrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)7, and transforming growth factor beta 1 (TGF- β 1) as potential markers of CKD-related complications such as tubular damage and fibrosis. Methods: Forty-one pre-dialysis children with CKD Stages 3–5 and 23 age-matched controls were enrolled in the study. The serum and urine concentrations of analysed parameters were assessed by an enzyme-linked immunosorbent assay test.
Cells within all multicellular organisms are surrounded by a multi-component structure, called the extracellularmatrix (ECM). The ECM is a dynamic network composed of several proteins (glycoproteins; collagens; proteoglicans like perlecan and others) synthesized and organized by the cells. This network can exist in different forms: as intersticial matrices (like the one of the connective tissue, a porous structure that allows cell movements and support) and as pericellular matrices (like the basement membrane, also known as the basal lamina, a sheet- like structure that serves as a barrier), providing support to cells and tissues (Schwarzbauer, 1999; Yurchenco et al., 2004). As the ECM interacts with cells directly and serves as a reservoir for growth factors, it gives the positional and environmental information needed for cells to coordinate their (own) behaviour. Several studies have been describing the ECM as a key player in embryogenesis (reviewed by Zagris, 2001). From the large variety of ECM molecules, laminin and fibronectin glycoproteins are the most studied so far, and those are the ones studied in this thesis.
The cellular receptor for tenascins ap- pears to be the facultative chondroitin sul- fate proteoglycan receptor-type protein ty- rosine phosphatase ζ/ß (RPTPζ/ß). Two other chondroitin sulfate proteoglycans can also bind tenascin, neurocan and the soluble form of RPTPζ/ß, phosphacan (43). In early post- natal and adult cerebellum, neurocan and phosphacan show immunoreactivities that follow different developmental time courses. Neurocan is seen in the prospective white matter and in granule cells, Purkinje cells, and molecular layer, whereas phosphacan is associated with Bergmann glial fibers in the molecular layer and their cell bodies below the Purkinje cells (44). Besides the chon- droitin sulfate proteoglycans, tenascins also bind heparin through their fifth fibronectin type III domain, although it is not clear if this in vitro binding capacity is also valid for binding to heparan sulfates that would be found in vivo, and if this is somehow related to neuronal migration (45).