Sponge Geodia Cydonium Research Articles

Characterization of the receptor protein-tyrosine kinase gene from the marine sponge Geodia cydonium.

Cells are provided with well-defined receptor structures (signal receivers) which interact with their corresponding ligands (signal molecules) and initiate a signal transduction pathway resulting in a change of cellular behavior or metabolism (Stoddard et al. 1992). It is well established that cells from both eukaryotic protists (single-cell organisms) and from Metazoa (multicellular organisms) respond to signals emanating from the extracellular environment. The extracellular signals to which protists respond are mainly nutrients which diffuse to their surfaces, and in most cases cross the cell membrane. In addition, they are able to bind peptide hormones, e.g., insulin or adrenocorticotropic hormone, as in the unicellular Tetrahymena, by receptor-like structures (Kohidai et al. 1994). Based on experimental data obtained with Tetrahymena, it has been proposed that the survival of protists presupposes the operation of highly dynamic membrane structures capable of recognizing a variety of environmental signals, interactions which are stored in a form of “memory” and transmitted to the progeny generation (Csaba 1987, 1994). Hence, in unicellular eukaryotes, the membrane-bound receptor(s) have a nondetermined ability to recognize ligands and are initially not genetically programmed. In contrast, in Metazoa the receptors are genetically preprogrammed.

A galectin links the aggregation factor to cells in the sponge (Geodia cydonium) system.

The cDNA for the full-length lectin from the marine sponge Geodia cydonium was cloned. Analysis of the deduced aa sequence revealed that this lectin belongs to the group of galectins. The full-length galectin, which was obtained also in a recombinant form, has an M(r) of 20,877; in the processed form it is a 15 kDa polypeptide. The enriched aggregation factor from G.cydonium also was determined to contain, besides minimal amounts of the galectin, a 140 kDa polypeptide which is involved in cell-cell adhesion. Monoclonal antibodies have been raised against this protein; Fab' fragments prepared from them abolished cell-cell reaggregation. Cell reaggregation experiments revealed that the aggregation factor is an essential component in the aggregation process but it requires likewise the presence of the galectin. Antibodies against the galectin blocked the aggregation factor-mediated cell adhesion. A plasma membrane component was identified (a 29 kDa polypeptide) which interacted with the aggregation factor in the presence of galectin; binding could be blocked both by antibodies against the galectin as well as against the aggregation factor. Immunohistochemical analysis revealed that spherulous cells contain the galectin but not the aggregation factor. By laser scanning microscopy, it is shown that both the aggregation factor and the galectin are located at the rim of the cells. From these data we conclude that the G.cydonium aggregation factor binds to the cells via a galectin bridge.

Cloning of a heat-inducible biomarker, the cDNA encoding the 70 kDa heat shock protein, from the marine sponge Geodia cydonium:response to natural stressors

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 136:153-161 (1996) - doi:10.3354/meps136153 Cloning of a heat-inducible biomarker, the cDNA encoding the 70 kDa heat shock protein, from the marine sponge Geodia cydonium: response to natural stressors Koziol C, Wagner-Hülsmann C, Mikoc A, Gamulin V, Kruse M, Pancer Z, Schäcke H, Müller WEG The biomarker concept involves the use of biochemical, cellular and physiological parameters as screening tools in environmental surveillance. Stress proteins, such as heat shock proteins (HSPs), fulfill many of the requirements for being ideal candidates in a biomarker strategy for environmental monitoring. Sponges (Porifera) are one of the major phyla found in the marine hard-substrate benthos, both with respect to the number of species and biomass. However, only recently have genes from sponges been cloned. Here we describe the isolation of the cDNA encoding a heat shock protein of Mr (relative molecular weight) 70 kDa (HSP70). The cDNA HSP70 from the marine sponge Geodia cydonium has a length of 2.3 kb and encodes an AA sequence of Mr 72579. The sponge HSP70 displays characteristic features of the HSP70 family. The HSP70 protein is induced by natural stressors including changes in temperature as well as pH, as demonstrated by Western blot analysis. No response was observed after treating the samples with hypotonic or hypertonic conditions. Our results provide the first molecular evidence that HSP70 of sponges is a useful biomarker. Geodia cydonium . Sponges . Heat shock protein . HSP70 . Environmental stress . Biomarker Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 136. Publication date: June 06, 1996 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1996 Inter-Research.

The (2'-5')Oligoadenylate Synthetase is Present in the Lowest Multicellular Organisms, the Marine Sponges. Demonstration of the Existence and Identification of its Reaction Products

We have proved the presence of (2′-5′)oligoadenylates [(2′-5′)An] and oligoadenylate synthetase [(2′-5′)An synthetase] in the marine sponge Geodia cydonium. (2′-5′)An isolated from sponge crude extract competed with authentic (2′-5′)An for binding to polyclonal antiserum against (2′-5′)An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2′-5′)A oligomers. The biological activity of sponge (2′-5′)An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2′-5′)An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2′-5′)An synthetase from sponge extract binds to poly(I) · poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43-kDa (2′-5′)An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2′-5′)An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control.

The (2′‐5′)Oligoadenylate Synthetase is Present in the Lowest Multicellular Organisms, the Marine Sponges

We have proved the presence of (2'-5')oligoadenylates [(2'-5')An] and oligoadenylate synthetase [(2'-5')An synthetase] in the marine sponge Geodia cydonium. (2'-5')An isolated from sponge crude extract competed with authentic (2'-5')An for binding to polyclonal antiserum against (2'-5')An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2'-5')A oligomers. The biological activity of sponge (2'-5')An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2'-5')An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2'-5')An synthetase from sponge extract binds to poly(I).poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43-kDa (2'-5')An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2'-5')An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control.

Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes — if present — are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomain-like sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals.

Phylogenetic relationship of ubiquitin repeats in the polyubiquitin gene from the marine sponge Geodia cydonium

Ubiquitin is a 76-residue protein which is highly conserved among eukaryotes. Sponge (Porifera) ubiquitin, isolated from Geodia cydonium, is encoded by a gene (termed GCUBI) with six repeats, GCUBI-1 to GCUBI-6. All repeat units encode the same protein (with one exception: GCUBI-4 encodes ubiquitin with a change of Leu to Val at position 71). On the nt level the sequences of the six repeats differ considerably. All changes (except in GCUBI-4) are silent substitutions, which do not affect the protein structure. However, there is one major difference between the repeats: Codons from both codon families (TCN and AGPy) are simultaneously used for the serine at position 65. Using this characteristic the repeats were divided into two groups: Group I: GCUBI-1,3 (TCT codon) and GCUBI-5 (TCC); Group II: GCUBI-2,4,6 (AGC codon). Mutational analysis suggests that the sponge polyubiquitin gene evolved from an ancestral monoubiquitin gene by gene duplication and successive tandem duplications. The ancestral monoubiquitin gene most probably coded for threonine (ACC) at position 65. The first event, duplication of the monoubiquitin gene, happened some 110 million years ago. Since sponges from the genus Geodia are known from the Cretaceous (145 million) to recent time, it is very likely that all events in the evolution of polyubiquitin gene occurred in the same genus. Alignment data of the "consensus" ubiquitin nt sequences of different animals (man to protozoa) reflect very well the established phylogenetic relationships of the chosen organisms and show that the sponge ubiquitin gene branched off first from the multicellular organisms.

Ubiquitin and ubiquitination in cells from the marine sponge Geodia cydonium.

Marine sponges, e.g. Geodia cydonium, have been intensively used to investigate the biochemical and molecular biological basis of cell-cell- and cell-matrix adhesion. It has been shown that a family of galactose-specific lectins, which are present in the extracellular space of G. cydonium, is a main component involved in cell-matrix adhesion in the sponge system. In the present study it is outlined that the purified 16-kDa lectin-1 binds to a 67-kDa membrane-associated protein. This lectin-binding protein undergoes mono- and diubiquitination after incubation of dissociated sponge cells with the homologous aggregation factor (AF), a molecule involved in cell-cell adhesion. The gene coding for polyubiquitin was characterized and found to be composed of three tandem repeat building blocks. Northern analysis indicated the presence of only one type of ubiquitin-specific mRNA (1.65 kb). The level of this transcript increased by 10-fold after incubation of the dissociated cells with AF for 8 h; in contrast, lectin-1 caused only a small effect on the steady-state level of ubiquitin mRNA. These data indicate that the expression of the polyubiquitin gene is directly or indirectly regulated by the AF and suggest that ubiquitination might be a process which controls the function of the membrane-associated lectin-binding protein during matrix-cell adhesion.

Cloning of the polyubiquitin cDNA from the marine sponge Geodia Cydonium and its preferential expression during reaggregation of cells

Ubiquitination of proteins is a critical step in the controlled degradation process of many polypeptides. Here we show that sponges, the simplest multicellular group of eukaryotic organisms, are also equipped with the ubiquitin pathway. The polyubiquitin cDNA was isolated and characterized from the marine sponge Geodia cydonium. The open reading frame contains six ubiquitin moieties, which are lined up head to tail without spacers. A comparison of the predicted amino acid sequence of the six sponge ubiquitin-coding units with those from other organisms revealed a high degree of homology (> 93%). The ubiquitin gene is expressed to almost the same extent in the two main compartments of the sponge, the cortex and the medulla. However, only in the cortex are detectable amounts of the ubiquitin protein synthesized. The ubiquitin protein isolated from the sponge organism was found to initiate protein degradation in the heterologous reticulocyte system in the same manner as bovine ubiquitin. In vitro studies with dissociated sponge cells revealed that the homologous aggregation factor causes (i) a strong increase in the steady-state level of mRNA coding for ubiquitin and (ii) a drastic increase in ubiquitin protein synthesis, while the homologous lectin failed to display that effect in isolated cells. These data suggest that ubiquitin may play a role in sponge morphogenesis.

Modulation of organotin-induced apoptosis by the water pollutant methyl mercury in a human lymphoblastoid tumor cell line and a marine sponge

Apoptosis IS a special form of cell death in which the cells act~vely participate in the process of dying. We found that the water pollutants tnbutylt~n (> 1 PM) and methyl mercury (MeHg; at concentrations of >3 FM) induce apoptosls In human T-lymphoblastoid CEM cells and in tlssue of the marlne sponge Geodia cydonium At concentrations of >5 yM. MeHg causes alkaline-labile sites in DNA of CEM cells. At the lower dose of 0.3 FM, MeHg abolishes the tr~butyltln-induced apoptosis in both CEM cells and sponge tissue. Incubation of sponge t~ssue w ~ t h 3 yM of tnbutyltin Induces not only apoptosis but also an increased expression of heat shock protein-70.

S-type lectins occur also in invertebrates: high conservation of the carbohydrate recognition domain in the lectin genes from the marine sponge Geodia cydonium.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various species following their pairwise comparison establishes a dendrogram, which reveals that the sponge lectin could be considered as the ancestor for vertebrate S-type lectins.

Immunological and biological identification of tumour necrosis-like factor in sponges: Endotoxin that mediates necrosis formation in xenografts

Xenografts of the sponge Geodia cydonium in its closely related species G. rovinjensis resulted in a rapid rejection of the graft within a period of 5 days. We identified an immunoreactive tumour necrosis factor (TNF)-like activity in the xenograft ( M r of 30 000) two days after grafting. In-vivo injection of 5 μg human recombinant TNF-α induced cytotoxicity in sponge cells in the same pattern and time course as during natural xenograft rejection. Anti-TNF-α polyclonals were found to react with xenograft extracts, by Western blot analysis, as from day 2 after grafting. Using ELISA we detected the TNF-like activity from day 2 after grafting with peak levels at days 4 and 5, where the amount was 0.72 ng/μg tissue DNA. By day 1, gp27 (inhibitory aggregation factor) is already formed in the xenograft. In-vitro experiments on isolated G. cydonium cells showed that addition of purified gp27 induced the production of the TNF-like activity (up to 13.5 ng/ml). Evidence is presented that gp27 is a product of the gp180 lectin receptor. We conclude that gp27 induces TNF-like factor production, resulting in destruction and dissolution of the xenograft after 5 days.

Histochemical reactivity of the Geodia cydonium agglutinin (GCA) in human tissues.

We applied a peroxidase-antiperoxidase technique to study the distribution pattern and binding characteristics of the lectin from the marine sponge Geodia cydonium (Geodia cydonium agglutinin; GCA) in various human tissues. This lectin has been shown to possess a broad reactivity, but there was a distinct distribution of binding sites within the different organs. In the histochemical system GCA displayed no blood group specificity and labeled red blood cells, the vascular endothelium, and epithelial cells showing blood group antigen expression independent of the ABH blood group status. However, inhibition of GCA reactivity by simple sugars and complex carbohydrates demonstrated tissue-specific differences of lectin binding related to the ABH blood group status of the tissue and revealed information on the structural requirements of the histological lectin binding site. Tissues that totally lacked blood group antigens or that expressed only the H-antigen disclosed a GCA reactivity which was completely inhibited by lactose. In contrast, tissues that expressed blood group A- or blood group B-antigen exhibited a lactose-resistant lectin binding which was inhibited only by water-soluble blood group substance A from peptone A and by bovine glycophorin but not by other complex carbohydrates, including human glycophorin and human asialoglycophorin. Competitive inhibition studies in situ revealed that GCA binding was not inhibited by blood group type I/II carbohydrate sequence-specific lectins or by lectins with other sugar specificities. Inhibition by lactose of GCA binding to some histological sites indicates that the binding site consists of a beta-linked galactose-containing disaccharide. However, periodate oxidation of tissue sections had no effect on lectin binding, pointing to a subterminal location of the relevant sequence. The results obtained from inhibition studies with simple saccharides and complex carbohydrates in relation to the expression of ABH blood group antigens suggest a complex lectin combining site(s) in histological specimens. The lectin may possess either one binding site with a range of affinities for different carbohydrates (besides beta-linked disaccharides the GCA binding site accommodates to carbohydrate determinants carrying the blood group A or blood group B determinant), or may possess two different binding sites. Besides an acceptor site for beta-linked disaccharides, an additional binding site may exist accommodating to extended carbohydrate sequences related to A or B blood group structures. In conclusion, GCA represents a blood group-nonspecific lectin whose binding affinities are determined by the ABH blood group status of the tissue.

Inhibition of aggregation-factor-induced ras gene expression in the sponge Geodia cydonium by detergent-polluted seawater a sensitive biological assay for low-level detergent pollution

Inhibition of aggregation-factor-induced ras gene expression in the sponge Geodia cydonium by detergent-polluted seawater a sensitive biological assay for low-level detergent pollution

CDNA structure and expression of calpactin, a peptide involved in Ca2+-dependent cell aggregation in sponges

Aggregation of cells of the marine sponge Geodia cydonium is mediated by an aggregation factor (AF) particle of Mr 1.3 X 10(8). It is now reported that the AF particle is associated with calpactin, which was ascribed a role in the cell-adhesion process. In order to identify the sequence similarity to other members of the lipocortin family, the cDNA of sponge calpactin was cloned and found to display an 80% sequence similarity to vertebrate calpactin II but only a 47% similarity to calpactin I. The calpactin gene, which contains the consensus sequence coding for the amino acids G-T-D-E, was expressed in Escherichia coli and subsequently purified to a 37000-Mr polypeptide. Both the p32 and the p37 are provided with approximately two Ca2+ ions/molecule and the property to bind to phospholipids. The dissociation constant (calpactin-Ca2+) was in the absence of phospholipids in the range 500-700 microM-Ca2+ but in their presence about 20-30 microM-Ca2+. On the basis of (i) inhibition studies with antibodies to calelectrin and (ii) competition experiments with soluble phospholipids (both chemically defined as well as total homologous membrane lipids) we conclude that the AF-associated calpactin and plasma-membrane-bound phospholipid(s) are involved in cell-cell aggregation in sponges.

Regulated expression and phosphorylation of the 23–26‐kDa ras protein in the sponge Geodia cydonium

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.

Alterations inras-gene expression and intracellular distribution of protein kinase C in the spongeGeodia cydonium in response to marine pollution

The siliceous spongeGeodia cydonium Jameson was used to study the influence of pollution in marine environments on selected parameters of the intracellular signal transduction pathway. The parameters chosen were: intracellular distribution of protein kinase C (PK-C),ras-gene expression and DNA polymerasealpha (DNA Polalpha) activity. Both PK-C andras-gene product (ras-protein) have previously been established to be key molecules in the intracellular signalling pathway in sponges; increased level ofras-protein mediates events following sponge cell-cell contact. Three unpolluted and three polluted sites in the off-shore seawater around Rovinj (Yugoslavia) were selected for the study in 1989. The state of pollution of these sites has been well-defined in a series of previous studies (1976 to 1989). Transplantation of regenerating sponge cubes ofG. cydonium to the polluted sites resulted in pronounced changes in the parameters chosen, compared to controls exposed to unpolluted environments. Expression ofras gene was increased by three- to five-fold after exposure of regenerating sponge tissue to the impacted sites. At the site with the highest pollutional load,ras mRNA level was about 50% of that at the reference sites. In parallel experiments it was established that, in response to pollution, a translocation of PK-C from the cytosolic to membrane fraction occurred. At the most impacted site, most of the enzyme activity was cytosolic. DNA Polalpha-activity, as a measure of sponge cell proliferation, decreased in a pollutioncorrelated manner. Our results indicate that the intracellular signalling system within sponge cells is activated in response to moderate pollution but is depressed in heavily polluted environments.

Intracellular signal transduction pathways in sponges

Sponges are the lowest multicellular eukaryotic organisms. Due to the relatively low specialization, and concomitantly the high differentiation and dedifferentiation potency of their cells, the sponge cell system has proven to be a useful model to study the mechanism of cell-cell adhesion on molecular levels. Results of detailed biochemical and cell biological studies with the main cell adhesion molecules, the aggregation factor (AF) and the aggregation receptor, led to the formation of the modulation theory of cell adhesion. The events of cell adhesion are contigent on a multiplicity of precisely coordinated intracellular signal transduction pathways. Using the marine sponge Geodia cydonium we showed that during the initial phase of cell-cell contact the AF causes a rapid stimulation of the phosphatidylinositol pathway, resulting in an activation of protein kinase C and a subsequent phosphorylation of DNA topoisomerase II. As one consequence of these processes, the cells undergo a phase of high DNA synthesis. However, at later stages, the AF loses its mitogenic activity; this function is then taken over by the matrix lectin. During this switch, the lectin receptor associates in the plasma membrane with the ras oncogene product. The description of these processes is subject of this review article.

Demonstration of an endocrine signaling circuit for insulin in the sponge Geodia cydonium.

The existence of an insulin-mediated cell-to-cell signaling in the sponge Geodia cydonium is demonstrated in this study by molecular biological and immunological techniques. The sequence of a sponge cDNA clone encoding preproinsulin was analyzed for the first time and determined to comprise a high homology to human preproinsulin (60-80% homology). The predicted polypeptide of preproinsulin from sponge contains two disulfide bridges which link the A- to the B-chain. The intra-A chain disulfide bridge is absent. Applying immunological and electron microscopical techniques it is shown that insulin is produced in specialized cells (spherulous cells). Experimental evidence is presented which indicates that the sponge preproinsulin (predicted Mr 11,850) is processed to insulin (Mr 5600; B-chain, Mr 3700 and A-chain, Mr 1900). Plasma membranes of sponge cells are shown to be provided with an insulin-binding receptor composed of two molecules (Mr 104,000 and Mr 98,000). Heterologous insulin (from bovine pancreas) was found to stimulate gene expression in G. cydonium cells. It is concluded that sponges are provided with an endocrine signaling circuit: signaling cells (spherulous cells), hormone (insulin), and hormone receptor bearing target cells which respond to the hormone stimulus.

Directed migration of cells from the sponge Geodia cydonium

The migration behavior of cells from the sponge Geodia cydonium was studied in vitro, applying the ‘Tissue Culture Slide Chamber’ technique. The homologous lectin caused a directed cell migration with a maximal locomotory rate of 1.6 μm/min. Competition experiments using the solubilized lectin receptor (= antiaggregation receptor) revealed that the chemotactic ligand (= lectin) interacts directly with the lectin receptor which—in consequence—functions as the chemotactic receptor. The ability of the lectin to promote cell migration is abolished by coincubation with purified leucine aminopeptidase. Biochemical and immunochemical data revealed that this enzyme is present also on the surface of sponge cells. Furthermore, we present evidence that the chemotactic receptor (= anti-aggregation receptor) on the cell surface is, in an hitherto unknown manner, coupled with the intracellularly present actin filaments. From these data we conclude that the directed migration of Geodia cells is mediated by the interaction between the lectin (= chemotactic ligand) and the lectin receptor (= chemotactic receptor); it is very likely that also intracellular structural elements operate simultaneously and coordinately during cell migration.