A spectrin-like protein present on membranes of Amoeba proteus as studied with monoclonal antibodies

Monoclonal antibodies against a spectrin-like membrane-associated protein of xD amoebae. (Amoeba proteus) were used to determine the distribution of the protein and some of its characteristics. A total of 34 monoclonal antibodies recognizing different epitopes of the protein were obtained, of which seven stained cell membranes by indirect immunofluorescence. The spectrin-like protein had two subtypes of 225 and 220 kDa and several monoclonal antibodies cross-reacted with human erythrocyte spectrin when checked by indirect immunofluorescence staining and immunoblotting. Some of the antibodies also cross-reacted with antigens in HeLa cells and chick embryo fibroblasts. Polyclonal and monoclonal antibodies against Drosophila and human erythrocyte spectrins cross-reacted with the spectrin-like protein from amoebae. On the basis of these results, it was concluded that the protein is a spectrin. The protein was found on most cellular membranes of amoebae, including the plasma, nuclear, and phagosomal membranes, as well as symbiosome membranes.     

Development of host- and symbiont-specific monoclonal antibodies and confirmation of the origin of the symbiosome membrane in a cnidarian-dinoflagellate symbiosis

The "symbiosome membrane" as defined by Roth et al. (1988) is a single, host-derived membrane that surrounds an endosymbiotic organism, separating it from the cytoplasm of the host cell. However, in the case of cnidarian-dinoflagellate endosymbioses, clear identification of the symbiosome membrane is complicated by the fact that each algal symbiont is surrounded by multiple layers of apparent membrane. The origin and molecular nature of these membranes has been the subject of considerable debate in the literature. Here we report the development of host-specific (G12) and symbiont-specific (PC3) monoclonal antibodies that allow separation of the host and symbiont components of these multiple membranes. Using immunocytochemistry at both the light and the electron microscopic level, we present data supporting the conclusion that the definitive symbiosome membrane is a single, host-derived membrane, whereas the remainder of the underlying apparent membranes surrounding the algal cell are symbiont-derived. The potential for macromolecules associated with these membranes to act as cellular signals critical to recruiting symbionts and maintaining established symbioses is discussed.     

Macromolecules Involved in the Amoeba-Bacteria Symbiosis1

ABSTRACT. In the Amoeba-bacteria symbiosis, rod-shaped Gram-negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host-symbiont interactions, although their respective roles are not yet fully known. The symbiont-derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29-kDa protein was most abundant in the host cytoplasm, while the 96-kDa protein and LPS were found mostly on the symbiosome membranes. The 67-kDa protein was a GroEL analog and stayed within the symbionts. The host-derived 43-kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225-kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29-kDa and 67-kDa proteins were cloned and sequenced. The 29-kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper-like motif, suggesting a possible DNA-binding function. The DNA sequence of the 67-kDa protein gene showed a 70% identity with heat-shock-protein genes of Escherichia coli and Coxiella burnetii.     

Proteomic analysis of symbiosome membranes in Cnidaria–dinoflagellate endosymbiosis

Symbiosomes are specific intracellular membrane‐bound vacuoles containing microalgae in a mutualistic Cnidaria (host)–dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin‐XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X‐100 soluble and insoluble fractions, were subjected to 2‐D SDS‐PAGE and identified by MS using an LC‐nano‐ESI‐MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti‐apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.     

Characterization of Myosin Heavy Chain and Its Gene in Amoeba proteus

ABSTRACT Monoclonal antibodies against the myosin heavy chain of Amoeba proteus were obtained and used to localize myosin inside amoebae and to clone cDNAs encoding myosin. Myosin was found throughout the amoeba cytoplasm but was more concentrated in the ectoplasmic regions as determined by indirect immunofluorescence microscopy. In symbiont-bearing xD amoebae, myosin was also found on the symbiosome membranes, as checked by indirect immunofluorescence microscopy and by immunoelectron microscopy. The open reading frame of a cloned myosin cDNA contained 6,414 nucleotides, coding for a polypeptide of 2,138 amino acids. While the amino-acid sequence of the globular head region of amoeba's myosin had a high degree of similarity with that of myosins from various organisms, the tail region building a coiled-coil structure did not show a significant sequence similarity. There appeared to be at least three different isoforms of myosins in amoebae, with closely related amino acids in the globular head region.     

Cytoplasmic membrane systems involved in bacterium release into soybean nodule cells as studied with two Bradyrhizobium japonicum mutant strains

Two Bradyrhizobium japonicum, Tn5-induced, mutant strains, ML126 and ML150, were studied. Both induce host cell division to form normal-sized nodules that do not fix nitrogen and whose cells have very few bacteroids (Bar-). Early-infection (15 days post infection) cells have much endoplasmic reticulum (ER), numerous Golgi bodies, and large vacuoles that are probably secondary lysosomes. Later the cytoplasm of the host cells of both are dominated by hundreds of vesicles containing only finely fibrous material and that appear to originate by the degradation of the cell walls of the infection threads; they have been named "infection-thread wall degradation vesicles" (IWDV). Phosphotungstic acid-chromic acid (PACA) staining of thin sections shows that IWDV membranes and the plasma membranes of both the cells and infection threads usually stain quite intensely, while the membranes of other cell organelles do not. The membranes of the few symbiosomes present in the mutants also stain with PACA. This evidence suggests that largely the host-cell plasma membrane gives rise to both the vesicle and symbiosome membranes in these mutants. In cells induced by both mutants, ER appears to be deficient, a finding suggesting that an ER-synthesis signal is involved in the normal release process, that ER synthesis is prerequisite to a normal volume of release, and that insufficient ER can impair symbiosome formation. In the mutant-induced infections, normal lysosomes develop and engulf both symbiosomes and cytoplasmic vesicles, but the retardation of this activity is the probable cause of the cytoplasm becoming overloaded with vesicles.     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

Resuscitation of amebae deprived of essential symbiotes: Micrurgical studies*

SYNOPSIS. The effect of depletion and restoration of obligatory bacterial endosymbiotes on Amoeba proteus strain xD was studied. Removal of the symbiotes by culturing the amebae at 26.5 C resulted in loss of viability of the host cells, indicating that this strain is dependent on its endosymbiotes for survival. Amebae depleted of bacteria could initially be resuscitated by injection of isolated symbiotes, but prolonged deprivation led to irreversible changes. Nuclei of aposymbiotic amebae were viable when transplanted into the cytoplasm of normal cells, but the symbiote-depleted cytoplasm of heat-treated amebae could not be resuscitated by renucleation. No immediate ultrastructural changes were detected in aposymbiotic amebae except for clumping of nucleoli. Thus it appears that the symbiote performs an essential function as a cytoplasmic constituent.     

BK virus-transformed inbred hamster brain cells. I. Status of the viral DNA and the association of BK virus early antigens with purified plasma membranes

Inbred LSH hamster brain cells were transformed in vitro by the GS strain of BK virus (BKV), and transplantable tumors classified as undifferentiated glioblastomas were induced in the syngeneic host. The viral status in the transformed cells, designated LSH-BR-BK, was established. About 46 genome equivalents per cell of viral DNA was detected, with the majority of sequences in a free form. The transformed cells expressed large quantities of tumor (T) antigen as well as surface (S) antigen as demonstrated by indirect immunofluorescence. Sixty-three percent of tumor-bearing hamsters produced high-titer antibodies against T, whereas 3 of 14 (21%) hamsters also produced antibodies against the BKV-specific S antigen. Furthermore, the relatedness of BKV early gene products, including T, S, and tumor-specific transplantation antigen, was established by the production of a rabbit antiserum against highly purified plasma membranes of LSH-BR-BK cells and by the induction of a BKV-specific tumor-specific transplantation antigen response by these plasma membranes in the syngeneic host.     

Detection with Monoclonal Antibodies of a 15-kDa Proteolipid in Both Presynaptic Plasma Membranes and Synaptic Vesicles in Torpedo Electric Organ

A protein, the mediatophore, has been purified from Torpedo electric organ presynaptic plasma membranes. This protein mediates the release of acetylcholine through artificial membranes when activated by calcium and is made up of 15-kDa proteolipid subunits. After immunization with purified delipidated mediatophore, monoclonal antibodies binding to the 15-kDa proteolipid band on Western blots of purified mediatophore were selected. A 15-kDa proteolipid antigen was also detected in cholinergic synaptic vesicles. Using an immunological assay, it was estimated that presynaptic plasma membranes and synaptic vesicles contain similar proportions of 15-kDa proteolipid antigen. Detection by immunofluorescence in the electric organ showed that only nerve endings were labeled. In electric lobes, the staining was associated with intracellular membranes of the electroneuron cell bodies and in axons. Nerve endings at Torpedo neuromuscular junctions were also labeled with anti-15-kDa proteolipid monoclonal antibodies.     

Localization of H+-ATPases in soybean root nodules

The localization of H⁺-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H⁺-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H⁺-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids. Received: 25 November 1998 / Accepted: 6 January 1999     

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.     

Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Lysosomal Membrane Proteins of Amoeba proteus, as Studied with Monoclonal Antibodies

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomoes from fusing with them.     

Lysosomal membrane proteins of Amoeba proteus, as studied with monoclonal antibodies.

Monoclonal antibodies were prepared against lysosomal membrane proteins of amoebae and used to follow lysosome-phagosome fusion after induced phagocytosis. The specificity of antibodies was checked by indirect immunofluorescence microscopy, immunoelectron microscopy, and localization of the antigen in subcellular fractions. The antibody-recognized proteins started to appear on the membranes of phagolysosomes about 5 min after phagocytosis as detected by indirect immunofluorescence, and the intensity of fluorescence increased for up to 1 h. Results of injection experiments in which purified antibodies had been injected into living cells and probed by indirect fluorescence indicated that the antigens were located on the cytoplasmic side of the lysosomal membranes. Lysosomes fuse with phagosomes on the one hand but not with non-fusible vesicles such as symbiosomes on the other. The results support the view that a membrane component(s) of non-fusible vesicles somehow prevents lysosomes from fusing with them.     

Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.     

Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways

During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.     

Immuno-electron microscopic localization of lipopolysaccharide antigens on ultrathin sections of Salmonella typhimurium.

Lipopolysaccharide antigens were demonstrated on ultrathin sections of styrene-embedded Salmonella typhimurium by direct postembedding staining with ferritin-labeled antibodies. The antigenicity, partially masked in the embedding process, could be satisfactorily recovered by treatment of ultrathin sections with nonspecific protease. As judged from the reaction site of the ferritin-labeled antibodies, the lipopolysaccharides were localized in two zones. The broader zone of densely distributed ferritin molecules was superimposed over the whole outer cellwall, and a smaller zone revealing antigenicity was found over the cell membrane, which strongly supports the concept that the latter is the site of synthesis of lipopolysaccharides. The well-defined labeled areas between these two antigenic zones may be the routes whereby the synthesized polysaccharide molecules reach the cell wall.