Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens- epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host- specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.     

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of (i) microfilament bundles containing proteins of contractile structures; (ii) microtubules; and (iii) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).     

Conspecificity of Elachista nigra and Elachista orbicularis (Elachistaceae, Phaeophyceae)

A taxonomic study of two brown algal species, Elachista nigra Takamatsu and Elachista orbicularis (Ohta) Skinner (Elachistaceae), was performed on the basis of morphological observations of field‐collected and laboratory cultured specimens from Japan (including their type localities) and molecular phylogenetic analyses. The two species had been distinguished by developmental patterns of paraphysis‐ and plurizoidangium‐bearing erect filaments, such filaments of E. nigra developing from wide erect filaments and those of E. orbicularis developing directly from basal prostrate filaments. However, many specimens investigated in the present study showed forms intermediate between these two patterns. Molecular phylogenetic analyses (including five additional elachistacean species) based on the internal transcribed spacer (ITS)2 region of the nuclear ribosomal RNA (nrRNA) gene showed a close relationship between all samples of E. nigra and E. orbicularis, and that the developmental patterns of paraphysis‐ and plurizoidangium‐bearing erect filaments were homoplasious. On the basis of these morphological and molecular data, E. orbicularis was reduced to synonymy with E. nigra. The ITS2 sequences of E. nigra were significantly different between samples from the Sea of Japan and those from the Pacific Ocean with several insertion/deletion and substitution mutations.     

Distribution of chitin/chitosan-like bioflocculant-producing potential in the genus Citrobacter

Some strains belonging to the genera Citrobacter and Enterobacter have been reported to produce chitin/chitosan-like bioflocculants (BFs) from acetate. In this study, to investigate the distribution of the BF-producing potential in the genus Citrobacter and to screen stably and highly BF-producing strains, we obtained 36 Citrobacter strains from different culture collection centers, which were distributed among seven species in the genus, and tested for the flocculating activities of their culture supernatants using a kaolin suspension method. As a result, 21 strains belonging to C. freundii (17 strains in 23 strains tested), C. braakii (two in two), C. youngae (one in one), and C. werkmanii (one in two) showed flocculating activity, but this ability was limited to cells grown on acetate. Gas chromatography/mass spectrometry (GC/MS) analysis of the hydrolysates from the BFs of five selected strains indicated that they consisted of glucosamine and/or N-acetylglucosamine, such as the chitin/chitosan-like BF (BF04) produced by Citrobacter sp. TKF04 (Fujita et al. J Biosci Bioeng 89: 40–46, 2000). Gel filtration chromatography using a high-performance liquid chromatography system revealed that the molecular weight ranges of these BFs varied, but the average sizes were all above 1.66?×?10⁶?Da.     

The mammalian iris-ciliary complex affects organization and synthesis of cytoskeletal proteins of organ and tissue cultured lens epithelial cells.

A water soluble growth inhibitor was isolated from the mammalian ocular iris-ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris-ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight proteins of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibition of their protein synthesis. Moreover, these tissue cultured lens epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens. The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H-leucine incorporation experiments showed that alpha-actin and alpha-tubulin synthesis is partially inhibited by IC factors in OCLE cells but vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells.     

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.     

A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Frequency tuning, latencies, and responses to frequency-modulated sweeps in the inferior colliculus of the echolocating bat, Eptesicus fuscus

Neurons in the inferior colliculus (IC) of the awake big brown bat, Eptesicus fuscus, were examined for joint frequency and latency response properties which could register the timing of the bat's frequency-modulated (FM) biosonar echoes. Best frequencies (BFs) range from 10 kHz to 100 kHz with 50% tuning widths mostly from 1 kHz to 8 kHz. Neurons respond with one discharge per 2-ms tone burst or FM stimulus at a characteristic latency in the range of 3–45 ms, with latency variability (SD) of 50 μs to 4–6 ms or more. BF distribution is related to biosonar signal structure. As observed previously, on a linear frequency scale BFs appear biased to lower frequencies, with 20–40 kHz overrepresented. However, on a hyperbolic frequency (linear period) scale BFs appear more uniformly distributed, with little overrepresentation. The cumulative proportion of BFs in FM₁ and FM₂ bands reconstructs a scaled version of the spectrogram of FM broadcasts. Correcting FM latencies for absolute BF latencies and BF time-in-sweep reveals a subset of IC cells which respond dynamically to the timing of their BFs in FM sweeps. Behaviorally, Eptesicus perceives echo delay and phase with microsecond or even submicrosecond accuracy and resolution, but even with use of phase-locked FM and tone-burst stimuli the cell-by-cell precision of IC time-frequency registration seems inadequate by itself to account for the temporal acuity exhibited by the bat. Accepted: 21 June 1997     

Cover Picture

The cover figure illustrates schematically the molecular linkages of cytoskeletal filaments to epithelial cell‐cell junctions. Microtubules are shown on the left, with schematic motors/cargoes, and connection to the zonula adhaerens and desmosomes. Actin filaments (top) and intermediate filaments (bottom) are shown on the right, with their connections to tight junctions, zonula adhaerens and desmosomes, respectively. See review by Sluysmans et al for identification of molecules.     

The expression of differentiation by chicken lens epithelium in in vitro cell culture

Dissociated cells of the lens epithelium of newly hatched chickens were cultured in vitro to investigate whether cells actively grown in culture retain their own differentive entiative traits to form lens fibers. After an exponential growth phase of the flattened epithelial cells, a number of “islets” of smaller epithelial cells with polygonal shape appeared. Along the periphery of these islets, the characteristic morphological change which leads to the formation of spherical bodies was observed. Electron microscopic observation showed the differentiation of lens fibers in these spherical bodies comparable to those in the lens in situ. Accumulation of δ-chrystallin was confirmed in such “lentoid” bodies. Outgrowth of the lens epithelial cells was maintained in in vitro culture up to about 50 days with several subculturings. The formation of lentoid bodies occurred in each subculture generation, which started from a homogeneous population of flattened epithelial cells. The present culture conditions permit the maintenance of such a population of cells that have a high growth potential and stably retains their differentiative trait to form lens fiber, even after repeated replication under in vitro conditions.     

Molecular and morphological diversity of Zygnema and Zygnemopsis (Zygnematophyceae,Streptophyta) from Svalbard (High Arctic)

ABSTRACT

Filamentous conjugating green microalgae (Zygnematophyceae, Streptophyta) belong to the most common primary producers in polar hydro-terrestrial environments such as meltwater streamlets and shallow pools. The mats formed by these organisms are mostly composed of sterile filaments with Zygnema morphology, but the extent of their diversity remains unknown. Traditional taxonomy of this group is based on reproductive morphology, but sexual reproduction (conjugation and formation of resistant zygospores) is very rare in extreme conditions. In the present study we gave the first record of zygospore formation in Svalbard field samples, and identified conjugating filaments as Zygnemopsis lamellata and Zygnema cf. calosporum. We applied molecular phylogeny to study genetic diversity of sterile Zygnema filaments from Svalbard in the High Arctic. Based on analysis of 143 rbcL sequences, we revealed a surprisingly high molecular diversity: 12 Arctic Zygnema genotypes and one Zygnemopsis genotype were found. In addition, we characterized individual Arctic genotypes based on cell width and chloroplast morphology using light and confocal laser scanning microscopy. Our findings highlight the importance of a molecular approach when working with sterile filamentous Zygnematophyceae, as hidden diversity might be very beneficial for adaptation to harsh environmental conditions, and experimental results could be misinterpreted when hidden diversity is neglected.     

Flagellar filaments of the deep-sea bacteria Idiomarina loihiensis belong to a family different from those of Salmonella typhimurium

The helical filaments of the bacterial flagella so far studied seem to be universal in the bacterial kingdom. Despite the variation in flagellin molecular masses, which range from 24 kDa to 62 kDa in different species, there are only two forms: either the so-called Normal (left-handed) or the Curly (right-handed). The Normal and Curly helical forms are asymmetric; the two characteristic helical parameters, which are the pitch and diameter, of Normal filaments are twice those of Curly filaments. Both the universality of these two helical forms and their asymmetry are biological puzzles. We found that the marine bacteria Idiomarina loihiensis have flagella with left-handed Curly-like filaments. Analysis of the polymorphic forms under different pH conditions showed that the Curly-like filaments are actually Normal filaments having a smaller pitch and diameter than those of Salmonella typhimurium. A minor modification of Calladine's model for a filament lattice can explain the variant helical forms. Pseudomonas aeruginosa filaments also belong to the family of I.loihiensis filaments. Thus, there are at least two families of flagella filaments.     

Development of the siphonous green alga Penicillus and the Espera state*

The thallus of Penicillus is composed of two filament types: axial filaments of indeterminate growth and laterals of determinate growth. In vegetative reproduction new plants arise from horizontal rhizoids. Four stages can be distinguished in development. In the primordium stage the tip of a rhizoid swells and forms a primordium. In the germling stage ascending and descending axial filaments arise from the primordium, the former grow into a fascicle and give rise to lateral saccate branches, each of which forms an ascending and a descending arm and branches further into rhizoid-like branchlets. Together these structures constitute the foundation of the stipe. At the same time the descending axial filaments elongate and become main rhizoidal filaments with lateral rhizoidal branchlets. In the early juvenile stage the stipe is formed. The elongating ascending axial filaments form a medulla while their laterals produce a cortex. In the late juvenile stage the axial filaments form the capitulum. The Espera state of Penicillus lacks a stipe since the ascending axial filaments do not join in a fascicle. Espera has been grown in laboratory cultures from Penicillus plants collected in the Caribbean region and also been found uncommonly in nature in this area. This state may be a response to environmental stress. The flattening of thalli and their orientation perpendicular to the direction of waves are discussed. A comparison of Penicillus and Codium indicates that at least two types of development exist in multiaxial Eusiphoniidae.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

Comparative floral structure and systematics in the balsaminoid clade including Balsaminaceae,Marcgraviaceae and Tetrameristaceae (Ericales)

Based on molecular phylogenetic studies, Balsaminaceae, Tetrameristaceae (including Pellicieraceae) and Marcgraviaceae form the strongly supported first branching clade in the asterid order Ericales. Marcgraviaceae and Tetrameristaceae were proposed to be closely related in pre‐molecular studies, but the systematic position of Balsaminaceae has been controversial for some time and a relationship with the other two families was never suggested in pre‐molecular/pre‐cladistic times. However, interfamilial relationships in the clade are still unclear because of conflicting phylogenetic hypotheses from molecular analyses. In order to assess the validity of these molecular hypotheses from a morphological point of view, the floral morphology, anatomy and histology of Balsaminaceae, Tetrameristaceae and Marcgraviaceae are comparatively studied in detail. In addition, earlier literature is reviewed. The monophyly of the balsaminoid clade is strongly supported by floral structure, and a series of potential floral synapomorphies is identified for the clade. Prominent features shared by the three families include broad and dorsiventrally flattened filaments, thread‐like structures lining the stomia of dehisced anthers, secretory inner morphological surfaces of the gynoecium, ovules intermediate between uni‐ and bitegmic, incompletely tenuinucellar ovules, fruits with persistent style and stigma, seeds lacking endosperm and several anatomical/histological traits. The families are also distinctive because the bracts and/or sepals are petaloid and nectariferous. Further, the floral structure supports a sister group relationship between Balsaminaceae and Tetrameristaceae rather than any of the other possible interfamilial relationships. These two families share a caducous calyx, post‐genital fusion/coherence of filaments and ovary surface, latrorse anther dehiscence, commissural carpel lobes and ovules with a thickened funiculus and a constricted chalazal region. The occurrence of these features in Ericales is discussed. Future structural studies in other ericalean lineages and additional molecular studies are needed to further test these features with respect to their systematic value for the balsaminoid clade. Some may turn out to be true synapomorphies, whereas others may be recognized as plesiomorphies, as they may be more widely spread in Ericales than currently thought. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 325–386.     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Binaural and frequency representation in the primary auditory cortex of the big brown bat, Eptesicus fuscus

This study examines the binaural and frequency representation in the primary auditory cortex (AC) of the big brown bat, Eptesicus fuscus, by using an ear-phone stimulation system. All 306 cortical neurons studied were excited by contralateral sound stimulation but they were either excited, inhibited or not affected by ipsilateral sound stimulation. These cortical neurons were columnarly organized according to their binaural and frequency-tuning properties. The excitation-excitation columns which occupy about 15% of the AC are mainly aggregated within an oval-shaped area of the central AC. The excitation-inhibition neurons and binaural neurons with mixed properties are distributed in the remaining 85% of the surrounding primary AC. Although the best frequency (BF) of these neurons shows a tendency to decrease from high to low along the anteroposterior axis of the primary AC, systematic variation in BF is not always consistent across the entire mapping area. In particular, BFs of cortical neurons isolated in the anterior AC vary quite unsystematically such that neurons with similar BFs are aggregated in isolated patches. Isofrequency and binaural columns are segregated into bands that intersect each other. Accepted: 13 August 1997     

Actin filament organization regulates the induction of lens cell differentiation and survival

The actin cytoskeleton has the unique capability of integrating signaling and structural elements to regulate cell function. We have examined the ability of actin stress fiber disassembly to induce lens cell differentiation and the role of actin filaments in promoting lens cell survival. Three-dimensional mapping of basal actin filaments in the intact lens revealed that stress fibers were disassembled just as lens epithelial cells initiated their differentiation in vivo. Experimental disassembly of actin stress fibers in cultured lens epithelial cells with either the ROCK inhibitor Y-27632, which destabilizes stress fibers, or the actin depolymerizing drug cytochalasin D induced expression of lens cell differentiation markers. Significantly, short-term disassembly of actin stress fibers in lens epithelial cells by cytochalasin D was sufficient to signal lens cell differentiation. As differentiation proceeds, lens fiber cells assemble actin into cortical filaments. Both the actin stress fibers in lens epithelial cells and the cortical actin filaments in lens fiber cells were found to be necessary for cell survival. Sustained cytochalasin D treatment of undifferentiated lens epithelial cells suppressed Bcl-2 expression and the cells ultimately succumbed to apoptotic cell death. Inhibition of Rac-dependent cortical actin organization induced apoptosis of differentiating lens fiber cells. Our results demonstrate that disassembly of actin stress fibers induced lens cell differentiation, and that actin filaments provide an essential survival signal to both lens epithelial cells and differentiating lens fiber cells.     

The cellular eye lens and crystallins of cubomedusan jellyfish

Summary The ultrastructure and major soluble proteins of the transparent eye lens of two cubomedusan jellyfish,Tripedalia cystophora andCarybdea marsupialis, have been examined. Each species has two complex eyes (one large and one small) on four sensory structures called rhopalia. The lenses consist of closely spaced cells with few organelles. The lens is situated next to the retina, with only an acellular layer separating it from the photoreceptors. SDS-PAGE showed that the large lens ofC. marsupialis has only two crystallin polypeptide bands (with molecular masses of approximately 20000 and 35000 daltons), while that ofT. cystophora has three bands (two with a molecular mass near 20000 daltons and one with a molecular mass near 35000 daltons). Interestingly, the small lens ofT. cystophora appears to be markedly deficient in or lack the lower molecular weight proteins. The crystallins behaved as monomeric proteins by FPLC and showed no immunological reaction with antisera of the major squid crystallin, chicken-crystallin or mouse-crystallin in western immunoblots. Very weak reactions were found with antimouse- and-crystallin sera. The 35000 dalton crystallin ofT. cystophora was purified and called J1-crystallin. It contained relatively high leucine (13%) and tyrosine (9%) and low methionine (2%). Several tryptic peptides were sequenced. Weak sequence similarities were found with- and-crystallins, which may account for some of the apparent weak immunological crossreactivity with these vertebrate crystallins. A polyclonal antiserum made in rabbits from a synthetic peptide of J1-crystallin reacted strongly with J1-crystallin ofT. cystophora andC. marsupialis in immunoblots; by contrast, no reaction was obtained with the lower molecular weight crystallins from these jellyfish, with the squid crystallin, or with any crystallins from the frog or human lens. Thus, despite the structural similarities between the cubomedusan, squid and vertebrate lenses, their crystallins appear very different.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - bp base pairs - PTC phenylisothiocyanate - FPLC fast phase liquid chromatography - NBRF National Biomedical Research Foundation A portion of this work was presented by Joram Piatigorsky at the First Hans Bloemendal Lecture in June 1988 in Nijmegen, The Netherlands