Laminin B1: Biological Overview | References
Gene name - Laminin B1
Cytological map position - 28C4-28D1
Function - ligand
Symbol - LanB1
FlyBase ID: FBgn0261800
Genetic map position - 2L: 7,811,440..7,820,774 [+]
Classification - Laminin-type epidermal growth factor-like domain
Cellular location - secreted
|Recent literature||Petley-Ragan, L.M., Ardiel, E.L., Rankin, C.H. and Auld, V.J. (2016). Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion. J Neurosci 36: 1151-1164. PubMed ID: 26818504
The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, β, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. This study demonstrates that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the β subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. It was found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits.
Laminins are heterotrimeric molecules found in all basement membranes. In mammals, they have been involved in diverse developmental processes, from gastrulation to tissue maintenance. The Drosophila genome encodes two laminin α chains (see Drosophila LanA), one β and one γ, which form two distinct laminin trimers. So far, only mutations affecting one or other trimer have been analysed. In order to study embryonic development in the complete absence of laminins, the gene encoding the sole lamininβ chain in Drosophila, LanB1 was mutated, so that no trimers can be made. LanB1 mutant embryos develop until the end of embryogenesis. Electron microscopy analysis of mutant embryos reveals that the basement membranes are absent and the remaining extracellular material appears disorganised and diffuse. Accordingly, abnormal accumulation of major basement membrane components, such as Collagen IV and Perlecan, is observed in mutant tissues. In addition, elimination of LanB1 prevents the normal morphogenesis of most organs and tissues, including the gut, trachea, muscles and nervous system. In spite of the above structural roles for laminins, these results unravel novel functions in cell adhesion, migration and rearrangement. It is proposed that while an early function of laminins in gastrulation is not conserved in Drosophila and mammals, their function in basement membrane assembly and organogenesis seems to be maintained throughout evolution (Urbano, 2009).
Basement membranes (BMs) are specialised layers of extracellular matrix (ECM) covering the basal side of all epithelia and endothelia, and surrounding muscles, peripheral nerves and other tissues. BMs provide mechanical stability and physical barriers between different cell types and are important for tissue morphogenesis in metazoans. They have been implicated in many processes such as cell differentiation, shape, adhesion, survival and migration (Urbano, 2009).
Even though the composition of BMs varies according to tissues and developmental stages, they are mainly composed of two meshworks formed by laminins and collagen IV. They also contain other ECM components, such as Nidogen and proteoglycans. Laminins are a family of large heterotrimeric glycoproteins composed of three non-identical chains. The laminin trimer forms a cross-shaped structure consisting in three short arms, each formed by a different chain, and a long arm composed of the three assembled coiled chains. Data from mammalian cell culture has shown that while the α subunit can be secreted alone as a monomer, secretion of the β and γ chains requires simultaneous expression of all three chains and their assembly into α-β-γ heterotrimers (Yurchenco, 1997). Once secreted, laminins form a meshwork by self-assembly through interactions between the three short arms. In developing tissues, laminin assembly also requires the long arm to be tethered to receptors on the cell surface, such as integrins and dystroglycan (Urbano, 2009).
Laminins are common to tissues of most multicellular metazoans and they are highly conserved across evolution. However, whereas invertebrates possess one to two laminin heterotrimers, mammals possess at least 15, which are formed through combinations of 5α, 4β and 3γ subunits (Aumailley, 2005). They differ with respect to their tissue distribution, presumably reflecting diverse biological functions. Thus, whereas null mutations in laminin α1 results in early embryonic lethality (day E7) (Miner, 2004), lack of laminin α2 and α3 chains causes, respectively, severe muscular dystrophy and skin blistering both in mice and in humans. Mice failing to express the laminin β1 or γ1 subunits lack embryonic BMs and do not survive beyond embryonic day 5.5 (E5.5), suggesting some type of compensation between different α subunits (Urbano, 2009).
The Drosophila genome encodes only four laminin chains: two α chains (α1,2 and α3,5), one β chain and one γ chain. These form two trimers, lamininA (α3,5; β1; γ1) and lamininW (α1,2; β1; γ1). The first laminin α chain described in Drosophila, α3,5 (encoded by Laminin A, LanA), is most similar to vertebrate α3 and β5 and is part of the lamininA trimer. Experiments in cell culture have shown that lamininA is likely to bind PS1 integrin (αPS1βPS) and not PS2 integrin (αPS2βPS). This is supported by the similarity of phenotypes of LanA mutant embryos and those lacking the PS1 integrin. The second laminin α chain described in Drosophila, α1,2 (encoded by wing blister, wb), is most similar to vertebrate α1 and α2 chains and is part of the lamininW trimer. α1,2 contains an RGD (Arg-Gly-Asp) motif, which is a recognition site for PS2 integrin. Indeed, experiments with Drosophila S2 cells in culture have shown that RGD-containing peptides derived from laminin α1,2 can serve as PS2 integrin ligands (Graner, 1998). This is further supported by studies showing that lamininW is not recruited to muscle attachment sites in embryos carrying a mutation in the αPS2 subunit affecting the RGD binding site (Devenport, 2007; Urbano, 2009 and references therein).
Only mutations in the α subunits have been described in Drosophila so far. Null mutations in the LanA gene result in embryonic lethality with defects in somatic muscles, dorsal vessel (heart) and endoderm. The α3,5 chain is also required for proper localisation of anteroposterior markers in the oocyte and for normal pathfinding of pioneer axons in the brain. In addition, hypomorphic mutants and trans-heteroallelic combinations of LanA give rise to adult escapers that have disorganised rhabdomeres and display vein defects and wing blistering. Mutations in wb have wing blisters and defects in the dorsal vessel, trachea, muscles and rhabdomeres. Mutations in the laminin β subunit (encoded by Laminin B1, LanB1) or β subunit (encoded by laminin B2, lanB2) have not been characterised (Urbano, 2009).
In this work, null mutations were isolated in LanB1, allowing the generation of embryos lacking all laminin function. These embryos develop until the end of embryogenesis, suggesting that in Drosophila, as it is the case in nematodes and contrary to mice, laminins are not required for early embryonic morphogenetic events. Analysis of LanB1 loss reveals that laminins are required for accumulation of major ECM components, such as Collagen IV and Perlecan (Trol), into BMs. Furthermore, in the absence of the β chain, BMs are absent and the remaining extracellular material appears disorganised and diffuse. In addition, the results reveal new functions for laminins in cell adhesion, migration and rearrangement, and identify laminins as essential regulators of the morphogenesis of most organs in Drosophila (Urbano, 2009).
The isolation of loss-of-function mutations in the single laminin β subunit encoded by the Drosophila genome has allowed a study laminin requirements during development. In absence of laminin β, other laminin subunits as well as major BMs components fail to assemble into BMs. This analysis reveals new functions for laminins in cellular adhesion, migration and rearrangement during organogenesis (Urbano, 2009).
Data from cell-culture experiments suggest that only laminin trimers are secreted extracellularly. The current model is that a transitional dimeric configuration composed of a β and γ chain is first assembled intracellularly before incorporation of an α chain allows secretion (Goto, 2001; Kumagai, 1997; Morita, 1985; Peters, 1985). This model predicts that in absence of the laminin β or γ subunits no functional laminin trimers could be exported. The experiments showing that in laminin β mutant embryos the laminin α3,5 and laminin α1,2 chains are not present at BMs or muscle attachment sites fully support this model (Urbano, 2009).
The network of collagen IV was thought to provide an initial scaffold that incorporates other BM components, including laminins, nidogens and perlecan. However, analysis of collagen IV mutants in mice and C. elegans has shown that collagen IV was in fact dispensable for deposition and initial assembly of BMs. Similarly, in Drosophila embryos lacking collagen IV or SPARC, a collagen IV interacting protein, the association of laminin and perlecan with cell surfaces was not affected until late embryogenesis (Martinek, 2008). However, when examined the other way around, genetic and developmental studies in the mouse and in C. elegans have demonstrated that laminins are essential for BM assembly in these two species. This study has shown, at both microscopic and ultrastructural levels, that this is also the case in Drosophila. Thus, the crucial role of laminins as a scaffold for recruitment of BM components is conserved throughout animal evolution (Urbano, 2009).
In contrast to BMs, this study found that laminins are not required for assembly of ECM components at the specialised matrix present at muscle attachment sites. This could be explained by the fact that assembly of this matrix clearly differs from assembly of BMs, as it is mainly mediated by cell-cell interactions. An alternative explanation might be that transmembrane receptors, such as integrins, could promote association of the tendon matrix to the cell surface independently of laminin networks. The findings that integrins become localised to muscle attachment sites in the absence of laminins and integrins are essential for lamininW recruitment (Devenport, 2007) support this idea (Urbano, 2009).
In mouse, laminin β1 is required for embryo implantation and gastrulation. Lamb1-/- embryos lack BMs and do not survive beyond embryonic day 5.5 (Miner, 2004). Similarly, in sea urchin embryos, injections with antibodies to laminin α chain inhibit gastrulation and spicule formation (Benson, 1999). However, RNAi inhibition of either β, γ or both α laminin genes in C. elegans did not impair embryonic development before the elongation stage (Kao, 2006), at which stage mutant embryos stopped developing and displayed severe defects in BM integrity and tissue development. These different requirements for laminins during early stages of embryonic development could be explained by considering that in sea urchins and amniotes the basal matrix forms shortly before gastrulation, whereas in nematodes, as well as in insects and amphibians, a basal lamina develops only at the end of gastrulation (Stern, 2004). An alternative explanation could be that laminins are required for epithelialisation and while in mammals this process precedes gastrulation, in nematodes, gastrulation precedes epithelialisation. In Drosophila mutations in either of the two laminin trimers result in late embryonic lethality. However, the role of laminins during gastrulation in Drosophila has remained an open question, as each trimer could compensate for the absence of the other. This study shows that removal of all laminins in Drosophila does not affect gastrulation, demonstrating that in Drosophila, and contrary to the mouse (Miner, 2004), the integrity of BMs is not crucial for this developmental event. Taken altogether, it is concluded that, although the late functions during organogenesis are well conserved, an early function for laminins is absent in Drosophila (Urbano, 2009).
During midgut morphogenesis, both the migration of endodermal cells and the subsequent transition to a polarised epithelium depend on the association of the endoderm with the visceral mesoderm. Laminins are deposited between these two cell layers, yet the overall morphogenesis of the midgut, including migration, midgut constriction, tube elongation and adhesion of the endoderm to the visceral mesoderm, was shown to occur normally in mutants for the Drosophila laminin α3,5 chain (Yarnitzky, 1995). This led to the proposal that these processes might be mediated by other substrates. This study has shown that this other substrate is the second laminin, since all these processes are affected in LanB1 mutant embryos. Whether this represents a unique function for the lamininW or requires both laminins awaits the analysis of these processes in embryos lacking just laminin α1,2 (Urbano, 2009).
Experiments in different model systems, such as the chick, axolotl and mouse, have demonstrated a role for laminins in the migration of different cell populations (Tzu, 2008). One of the best-studied processes is the migration of the neural crest, which appears to migrate in response to heterogeneity in the ECM that forms their migration substrate. Collectively, these studies propose that neural crest migration may be governed by the relative ratio of permissive ECM components, such as fibronectin and laminin, versus non-permissive ECM components, such as chondroitin sulphate proteoglycans. Other ECM molecules, such as vitronectin, perlecan and several collagen types, seem to play a neutral role in this process (Henderson, 1997; Perris, 2000). In Drosophila, despite the implication of integrins in most migratory processes during embryogenesis, a clear role for laminins in regulating cell migration has remained elusive. To date, only mutations in the laminin α1,2 were shown to result in gaps in the tracheal dorsal trunk (Martin, 1999). This study shows that laminins are required for all integrin-dependent migrations described so far, including that of the endoderm, macrophages, salivary glands and trachea visceral branches. These results identify the laminins as being either the key integrin ligands regulating cell migration during Drosophila embryogenesis, or essential for recruitment of key ligands into the migration substrate. In this new scenario, several questions now arise. What cells provide the laminins? Do other Drosophila ECM molecules, such as Collagen IV or Perlecan, permit or inhibit cell migration? Are other laminin receptors beside integrins involved in cell migration in Drosophila? This study shows that in the absence of laminins, other BM components, such as Collagen IV and Perlecan, are not deposited around the VNC, and macrophages no longer migrate along this path. Inhibition of haemocyte migration impairs ECM deposition around the VNC (Olofsson, 2005). Thus, as macrophages need matrix components to migrate and BM deposition around the VNC requires macrophage migration, it is tempting to speculate that macrophages might be able to deposit their own matrix molecules for migration, making them independent of the matrix of the environment. For example, human keratinocytes deposit laminin 332 to promote their linear migration (Urbano, 2009 and references therein).
Laminins can interact with different types of receptors, including integrins, α-dystroglycan, sulphated carbohydrates (sulphatides, heparin, heparan sulphates and HNK-1) and the Lutheran antigen (Yurchenco, 2004). Several of the defects seen in LanB1 mutants are remarkably similar to those reported for loss of integrin function, including defects in: adhesion between the wing surfaces; macrophage and tracheal cell migration; proventriculus morphogenesis, elongation and formation of constrictions during gut development; and adhesion between visceral mesoderm and endoderm (Bradley, 2003
The current results are consistent with the idea that in Drosophila, as in mouse, laminins play a central role in organising the specialised ECM present at BMs and that this may represent a first important step for BM formation (Li, 2002). The establishment of a laminin-based BM scaffold is then crucial for many different cellular processes governing morphogenesis of most organs and tissues. Future studies are needed to address how laminins can perform such different developmental functions, including strong adhesion between different layers but also weak adhesion to allow cell migration. The information derived from these studies should help to understand the pathology of diseases related to abnormal laminin functions (Urbano, 2009).
SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. It was previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. This study sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency was found to lead to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell autonomous accumulation of BL components around mutant cells that include Collagen IV (Col IV), Laminin and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. These data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV (Shahab, 2014).
The emergence of multicellular organisms was co-incident with the appearance of genes coding for extracellular matrix (ECM) molecules that gave rise to two major classes of ECMs: interstitial matrices and basal laminae (BL)/basement membranes. In contrast to vertebrate tissues where interstitial matrices predominate, BL are the principal ECMs in animals of lower phyla. Universal components of BLs include network-forming Collagen IV (Col IV), Laminin, Perlecan, and Nidogen, which are assembled into 2D sheet-like networks. In addition to serving as tissue boundaries and an adhesive substratum for cell anchoring and migration, BLs make diverse regulatory contributions to the development of tissues and organs (Hohenester, 2013; Shahab, 2014).
Col IV imparts tensile strength to BL and provides an anchoring substratum for cell adhesion, migration, and secreted signaling molecules. Much of what is known about Col IV is derived from vertebrate studies. Vertebrates express six Col IV α-chains [α1(ΙV)-α6(ΙV)] that are assembled in the endoplasmic reticulum into different combinations of heterotrimeric protomers. Upon secretion, the C-terminal globular domain of these trimeric protomers form head-to-head dimers Flexible non-helical interruptions separating collagenous domains of the protomers promote lateral associations during supramolecular assembly of 2D Col IV networks. Further contributing to the stability of these networks, the N-terminal globular domain of the heterotrimers form anti-parallel tetramers. As with fibril-forming collagens, purified Col IV protomers can self-assemble into polymeric networks. In contrast to vertebrates, the Drosophila genome codes for only two Col IV α-chains: Dcg 1/Cg25C and Viking (Vkg). The primary sources of BL components produced within Drosophila embryos and larvae are hemocytes and the fat body (Olofsson, 2005); however, how Col IV and the other BL components are assembled into a stereotypic 2D sheet of precise thickness is unknown (Shahab, 2014).
Previously studies have shown that SPARC (Secreted Protein, Acidic and Rich in Cysteine), a highly conserved matricellular glycoprotein, is a major component of embryonic Drosophila BL (Martinek, 2002; Martinek, 2008). SPARC, also known as osteonectin/ BM40, binds to fibril-forming collagens and Col IV via epitopes located within the C-terminal domain. The absence of interstitial matrices in Drosophila makes it an ideal developmental and genetic model to decipher the role of SPARC in BL assembly and maturation (Shahab, 2014).
Using imprecise P-element excision to generate a mutation/deletion of SPARC in Drosophila, a previous study reported decreased Col IV and BL stability and neural defects resulting in embryonic lethality in the absence of SPARC. However, attempts to rescue embryonic lethality by expressing exogenous SPARC were unsuccessful (Martinek, 2008), raising the possibility that aspects of this phenotype were due to a second site mutation on the 3rd chromosome. The present study, determined that both the neural phenotype and embryonic lethality reported previously, result from a disruption of the neurogenic gene, neutralized. The disruption of SPARC alone leads to larval lethality characterized by compromised fat body homeostasis. The fat body is crucial for development. It acts as the primary source of energy, and fat body together with hemocytes are the principle sources of BL components during larval development. Formed during embryonic development, the larval fat body is a bilateral, multi-lobed organ consisting of a monolayer of about 2,200 polygonal cells called adipocytes. The larval fat body is entirely surrounded by hemolymph, but does not directly interface with it owing to the presence of a BL that covers the entire surface of the fat body. The adipocytes within the fat body have no classical apical-basal polarity. Instead, cell-cell adhesion and shape is mediated by BL surrounding the adipocytes (Pastor-Pareja, 2011). This study reports that a reduction of SPARC leads to defective fat body BL assembly, inducing the resident polygonal adipocytes to round up and accumulate BL components within their microenvironment in a cell-autonomous manner. These findings define a pivotal role for SPARC in the proper assembly of BL surrounding the adipocytes of the Drosophila fat body (Shahab, 2014).
The results of this study demonstrate that loss or knockdown of SPARC expression in Drosophila result in arrest during larval development and disruption of fat body architecture and function. Based upon the SPARC mutation Df(3R)nm136, it was previously reported that loss of SPARC resulted in embryonic lethality associated with severe defects in nervous system development. This study now provide evidence that a second-site mutation present in the neuralized locus, a key regulator of Notch/Delta signalling, is the cause of the Df(3R)nm136 neural phenotype and embryonic lethality. Hence, SPARC is not required for nervous system development (Shahab, 2014).
The new Df(3R)nm136 H2AvD::GFP line, from which the neuralized mutation has been removed, demonstrates that loss of SPARC in Drosophila results in larval lethality and morphological changes of the fat body. The larval fat body is a multifunctional organ essential to fly development. Principle functions of the organ are nutrient storage and regulation of energy availability, functions that may become compromised in SPARC-deficient larvae. SPARC-deficient larvae appear transparent, which is consistent with reduced lipid or energy stores. While it is possible that knockdown of SPARC in hemocytes was responsible for the lethality and fat body morphological defects, knockdown of SPARC selectively within hemocytes using a hemolectin promoter did not result in larval lethality or a fat body phenotype, indicating that the phenotype reported in this study is due to loss of fat body SPARC expression. Moreover, larval lethality and the fat body phenotype of SPARC mutant larvae were rescued by a SPARC transgene that was expressed under the control of either endogenous SPARC or Col IV promoters (Shahab, 2014).
SPARC reduction led to a marked accumulation of BL components in the extracellular microenvironment of affected adipocytes. Temporal expression data from modENCODE indicate that maximum levels of SPARC and Col IV expression occur during the 1st and 2nd instar stages, with expression decreasing during the 3rd larval instar prior to pupariation. Consistent with the idea that SPARC effects are largely mediated prior to the late 3rd instar stage, knockdown of SPARC in 3rd instar had no impact on survival or fat body remodeling (Shahab, 2014).
Pastor-Pareja (2011) showed that knockdown of SPARC results in extracellular assembly of Col IV into thick fibers in the fat body, leading them to speculate that SPARC is required for Col IV secretion and solubility. However, the impact of SPARC knockdown on Col IV secretion, BL integrity, or adipocyte morphology was not addressed in that study. The current study suggests that SPARC deficiency does not prevent Col IV secretion. Consistent with the results of Pastor-Pareja (2011), this study shows extracellular accumulation of Col IV, suggestive of decreased solubility. Moreover, this study shows that Laminin, Perlecan, and Nidogen also accumulate at the surface of SPARC-deficient adipocytes, indicating that all BL components are affected by the loss or knockdown of SPARC (Shahab, 2014).
Biochemical studies have shown that SPARC binds to the triple-helical domains of purified invertebrate and vertebrate Col IV, an interaction that is mediated by two collagen-binding epitopes located in the C-terminal region of SPARC. Col IV is a primary regulator of cell shape and adhesion; thus, alterations in the availability or structure of Col IV fibrils impact cell morphology. Several studies have shown that SPARC has counter-adhesive activity in vitro that causes cells to detach from their substrate and round up. The current data appear paradoxical as loss of SPARC results in cell rounding but does not lead to adipocyte dissociation. However, the impact on cell shape in this instance is likely due to the dysregulation of Col IV polymerization and BL homeostasis, rather than directly to the effect of SPARC on cell-cell or cell-matrix interactions (Shahab, 2014).
A previous studies suggested that SPARC co-localizes with Col IV within secretory vesicles of adipocytes, but it remains to be determined whether SPARC and Col IV directly bind to one another intracellularly. Upon exocytosis, close proximity of SPARC with Col IV enables immediate physical association such that SPARC can regulate Col IV polymerization and sequester Col IV from its cellular receptors. Bradshaw (2009) demonstrated such a relationship between SPARC and Collagen I in mammalian cells. SPARC deficiency does not lead to an increase in intracellular Col IV, demonstrating that the impact of a lack of SPARC on Col IV assembly into BL likely occurs extracellularly. Upon secretion, SPARC may act to maintain solubility of Col IV, preventing it from immediately undergoing polymerization. In the absence of SPARC, Col IV release to the fat body extracellular space occurs; however, without SPARC to delay its polymerization, Col IV may rapidly assemble into a dense meshwork. Other ECM proteins, such as Laminin, Perlecan, and Nidogen, are synthesized and secreted; they encounter polymerized Col IV and are incorporated into the assembled structure as they would in a normal BL. This causes accumulation of multiple BL proteins on the surface of adipocytes. As ECM material accumulates, it promotes the rounding of the cells. The formation of a dense ECM meshwork likely impedes normal adipocyte function and could interfere with a variety of physiological processes such as feeding behavior and energy metabolism (Shahab, 2014).
In light of the diffusible nature of SPARC, the finding of a cell-autonomous phenotype with fat body SPARC knockdown clones was unexpected. The failure of SPARC secreted from adjacent wild-type adipocytes to compensate for the lack of production by SPARC-deficient cell clones indicates that SPARC was not able to diffuse across the BL in sufficient quantities. To date, no study that has addressed the ability of SPARC to diffuse across the BL, but the current data raise the possibility of a charge-dependent barrier that retains SPARC within the microenvironment of a cell. Alternatively, the more immediate interaction of SPARC with Col IV afforded by their intracellular co-localization may be required to effectively prevent premature polymerization of Col IV. Hence, an intracellular interaction between SPARC and Col IV may be required to regulate the kinetics of Col IV polymerization immediately upon its secretion (Shahab, 2014).
SPARC may also regulate BL deposition and remodelling through cell surface receptors. Expression of the cell-matrix adhesion molecules Dg and the βPS integrin subunit was observed on the plasma membrane of wild-type adipocytes. RNAi knockdown of SPARC did not alter the expression or localization of either of these transmembrane receptors in fat body cells indicating that it is unlikely that ECM accumulation around SPARC mutant adipocytes is associated with dysregulation of ECM receptors. However, the possibility that the interaction of BL components with these ECM receptors may have been affected cannot be excluded (Shahab, 2014).
Randomly distributed pits were observed on the surface of adipocytes, which increased in number with the knockdown of SPARC. However, the majority of the pits associated with a SPARC knockdown exhibited thickened circumferential borders underlaid by intracellular lipid-like vesicles. It is conceivable that the pits represent sites of lipid exocytosis. However, preliminary data indicates that the knockdown of SPARC does not affect protein or vesicular endocytosis and exocytosis. Moreover, differences in lipid content between wild-type and SPARC-deficient adipocytes were not observed. Hence the molecular basis of the dramatic difference in the surface topography between wild-type and SPARC-deficient adipocytes remains unknown (Shahab, 2014).
Analysis of the evolutionary history of SPARC revealed a conservation of the collagen-binding epitopes from cnidarians to mammals, which enable SPARC to bind to fibril-forming and network-forming Col IV. While SPARC-null mice develop normally, ultrastructural analysis revealed that interstitial Col IV fibrils are less abundant, smaller and more uniform in size, resulting in fibrils with decreased tensile strength. Biochemical studies indicate that SPARC increases the length of the first stage/lag phase of collagen fibrillogenesis by decreasing the rate of nucleation (Bradshaw, 2009). SPARC is also concentrated in the basal laminae of the nematode C. elegans. RNAi knockdown of SPARC leads to larval lethality for a large percentage of the progeny with a deficiency in gut granules and reduction in body size (Fitzgerald, 1998). It remains to be determined if aberrations in BL lamina assembly is the underlying cause of the phenotype (Shahab, 2014).
Hence, these findings support an emerging concept of SPARC as a critical extracellular collagen chaperone. A detrimental loss of BL homeostasis is evident in the absence of SPARC. The evolutionary conservation of SPARC parallels the advent of BL in multi-cellular organisms, indicating that this chaperone activity of SPARC is important for the maintenance of ECM homeostasis in all metazoans (Shahab, 2014).
Search PubMed for articles about Drosophila Laminin
Aumailley M., et al. (2005). A simplified laminin nomenclature. Matrix Biol. 24: 326-332. PubMed ID: 15979864
Benson S., et al. (1999). Developmental characterization of the gene for laminin alpha-chain in sea urchin embryos. Mech. Dev. 81: 37-49. PubMed ID: 10330483
Bradley, P. L., Myat, M. M., Comeaux, C. A. and Andrew, D. J. (2003). Posterior migration of the salivary gland requires an intact visceral mesoderm and integrin function. Dev. Biol. 257: 249-262. PubMed ID: 12729556
Bradshaw, A. D., Baicu, C. F., Rentz, T. J., Van Laer, A. O., Boggs, J., Lacy, J. M. and Zile, M. R. (2009). Pressure overload-induced alterations in fibrillar collagen content and myocardial diastolic function: role of secreted protein acidic and rich in cysteine (SPARC) in post-synthetic procollagen processing. Circulation 119: 269-280. PubMed ID: 19118257
Chanana, B., et al. (2007). AlphaPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin. Mech. Dev. 124: 463-475. PubMed ID: 17482800
Devenport D., et al. (2007). Mutations in the Drosophila alphaPS2 integrin subunit uncover new features of adhesion site assembly. Dev. Biol. 308: 294-308. PubMed ID: 17618618
Fitzgerald, M. C. and Schwarzbauer, J. E. (1998). Importance of the basement membrane protein SPARC for viability and fertility in Caenorhabditis elegans. Curr Biol 8: 1285-1288. PubMed ID: 9822581
Goto, A., Aoki, M., Ichihara, S. and Kitagawa Y. (2001). alpha-, beta- or gamma-chain-specific RNA interference of laminin assembly in Drosophila Kc167 cells. Biochem. J. 360: 167-172. PubMed ID: 11696004
Graner M. W., et al. (1998). Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2. J. Biol. Chem. 273: 18235-18241. PubMed ID: 9660786
Hohenester, E. and Yurchenco, P. D. (2013). Laminins in basement membrane assembly. Cell Adh Migr 7: 56-63. PubMed ID: 23076216
Henderson D. J. and Copp A. J. (1997). Role of the extracellular matrix in neural crest cell migration. J. Anat. 191: 507-515. PubMed ID: 9449070
Kao G., et al. (2006). The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans. Dev. Biol. 290: 211-219. PubMed ID: 16376872
Kumagai C., Kadowaki T. and Kitagawa Y. (1997). Disulfide-bonding between Drosophila laminin beta and gamma chains is essential for alpha chain to form alpha betagamma trimer. FEBS Lett. 412: 211-216. PubMed ID: 9257722
Li, S., et al. (2002). Matrix assembly, regulation, and survival functions of laminin and its receptors in embryonic stem cell differentiation. J. Cell Biol. 157: 1279-1290. PubMed ID: 12082085
Martin D., et al. (1999). wing blister, a new Drosophila Laminin a chain required for cell adhesion and migration during embryonic and imaginal development. J. Cell Biol. 145: 191-201. PubMed ID: 10189378
Martinek, N., et al. (2008). Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos. J. Cell Sci. 121: 1671-1680. PubMed ID: 18445681
Martin-Bermudo, M. D., Alvarez-Garcia I. and Brown N. H. (1999). Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions. Development 126: 5161-5169. PubMed ID: 10529432
Martinek, N., Zou, R., Berg, M., Sodek, J. and Ringuette, M. (2002). Evolutionary conservation and association of SPARC with the basal lamina in Drosophila. Dev Genes Evol 212: 124-133. PubMed ID: 11976950
Martinek, N., Shahab, J., Saathoff, M. and Ringuette, M. (2008). Haemocyte-derived SPARC is required for collagen-IV-dependent stability of basal laminae in Drosophila embryos. J Cell Sci 121: 1671-1680. PubMed ID: 18445681
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date revised: 25 March 2015
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