Possible roles of actin and myosin during anaphase chromosome movements in locust spermatocytes

Summary. We tested whether the mechanisms of chromosome movement during anaphase in locust (Locusta migratoria L.) spermatocytes might be similar to those described for crane-fly spermatocytes. Actin and myosin have been implicated in anaphase chromosome movements in crane-fly spermatocytes, as indicated by the effects of inhibitors and by the localisations of actin and myosin in spindles. In this study, we tested whether locust spermatocyte spindles also utilise actin and myosin, and whether actin is involved in microtubule flux. Living locust spermatocytes were treated with inhibitors of actin (latrunculin B and cytochalasin D), myosin (BDM), or myosin phosphorylation (Y-27632 and ML-7). We added drugs (individually) during anaphase. Actin inhibitors alter anaphase: chromosomes either completely stop moving, slow, or sometimes accelerate. The myosin inhibitor, BDM, also alters anaphase: in most cases, the chromosomes drastically slow or stop. ML-7, an inhibitor of MLCK, causes chromosomes to stop, slow, or sometimes accelerate, similar to actin inhibitors. Y-27632, an inhibitor of Rho-kinase, drastically slows or stops anaphase chromosome movements. The effects of the drugs on anaphase movement are reversible: most of the half-bivalents resumed movement at normal speed after these drugs were washed out. Actin and myosin were present in the spindles in locations consistent with their possible involvement in force production. Microtubule flux along kinetochore fibres is an actin-dependent process, since LatB completely removes or drastically reduces the gap in microtubule acetylation at the kinetochore. These results suggest that actin and myosin are involved in anaphase chromosome movements in locust spermatocytes. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Cytochalasin induces abnormal anaphase in crane-fly spermatocytes and causes altered distribution of actin and centromeric antigens

Inhibition of cytokinesis by cytochalasins without an effect on karyokinesis has been demonstrated in several types of cells. We report here that treating crane-fly spermatocytes with cytochalasins at concentrations (10 M CE, 100 M CD, and 200 CB) in excess of that needed to inhibit cell division induces one or more half-bivalents to lag at anaphase during the first meiotic division. The behavior of the laggards is similar to that of maloriented half-bivalents. Following treatment at these concentrations, probing with rhodamine-phalloidin or bodipy-phallacidin reveals loss of filamentous actin from the poles and its appearance in the spindle, predominantly in regions where centromeres and kinetochores are normally found. When either N350 anti-actin monoclonal antibody or rhodamine DNase I was used to probe for actin in cytochalasin-treated cells, a similar redistribution of actin was observed. CD and CE treatments alter the pattern of fluorescence at centromere/kinetochore regions after staining with scleroderma CREST serum: CREST-positive structures become broader, with spikes extending from them toward the pole; in addition, some strands of CREST fluorescence appear that are apparently extraneous, and not associated with chromosomes. Probes for actin yield staining patterns in centromere/kinetochore regions that match closely the cytochalasin-altered pattern of CREST staining. Our finding of actin in the vicinity of kinetochores under conditions that result in abnormal chromosome behavior raises numerous questions about the possible role(s) of actin in meiosis, particularly in chromosome orientation.Abbreviations CREST calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia by W.C. Earnshaw     

Effects of anti-myosin drugs on anaphase chromosome movement and cytokinesis in crane-fly primary spermatocytes.

To investigate whether myosin is involved in crane-fly primary spermatocyte division, we studied the effects of myosin inhibitors on chromosome movement and on cytokinesis. With respect to chromosome movement, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime (BDM) added during autosomal anaphase reversibly perturbed the movements of all autosomes: autosomes stopped, slowed, or moved backwards during treatment. BDM added before anaphase onset altered chromosome movement less than when BDM was added during anaphase: chromosome movements only rarely were stopped. They often were normal initially and then, if altered at all, were slowed. To confirm that the effects of BDM were due to myosin inhibition, we treated cells with ML-7, a drug that inhibits myosin light chain kinase (MLCK), an enzyme necessary to activate myosin. ML-7 affected anaphase movement only when added in early prometaphase: this treatment prevented chromosome attachment to the spindle. We treated cells with H-7 as a control for possible non-myosin effects of ML-7. H-7, which has a lower affinity than ML-7 for MLCK but a higher affinity than ML-7 for other potential targets, had no effect. These data confirm that the BDM effect is on myosin and indicate that the myosin used for chromosome movement is activated near the start of prometaphase. With respect to cytokinesis, BDM did not block furrow initiation but did block subsequent contraction of the contractile ring. When BDM was added after initiation of the furrow, the contractile ring either stalled or relaxed. ML-7 blocked contractile ring contraction when added at all stages after autosomal anaphase onset, including when added during cytokinesis. H-7 had no effect. These results confirm that the effects of BDM are on myosin and indicate that the myosin used for cytokinesis is activated starting from autosomal anaphase and continuing throughout cytokinesis.     

Redundant mechanisms for anaphase chromosome movements: crane-fly spermatocyte spindles normally use actin filaments but also can function without them

Summary. Actin inhibitors block or slow anaphase chromosome movements in crane-fly spermatocytes, but stopping of movement is only temporary; we assumed that cells adapt to loss of actin by switching to mechanism(s) involving only microtubules. To test this, we produced actin-filament-free spindles: we added latrunculin B during prometaphase, 9–80 min before anaphase, after which chromosomes generally moved normally during anaphase. We confirmed the absence of actin filaments by staining with fluorescent phalloidin and by showing that cytochalasin D had no effect on chromosome movement. Thus, actin filaments are involved in normal anaphase movements, but in vivo, spindles nonetheless can function normally without them. We tested whether chromosome movements in actin-filament-free spindles arise via microtubules by challenging such spindles with anti-myosin drugs. Y-27632 and BDM (2,3-butanedione monoxime), inhibitors that affect myosin at different regulatory levels, blocked chromosome movement in normal spindles and in actin-filament-free spindles. We tested whether BDM has side effects on microtubule motors. BDM had no effect on ciliary and sperm motility or on ATPase activity of isolated ciliary axonemes, and thus it does not directly block dynein. Nor does it block kinesin, assayed by a microtubule sliding assay. BDM could conceivably indirectly affect these microtubule motors, though it is unlikely that it would have the same side effect on the motors as Y-27632. Since BDM and Y-27632 both affect chromosome movement in the same way, it would seem that both affect spindle myosin; this suggests that spindle myosin interacts with kinetochore microtubules, either directly or via an intermediate component. Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s00709-005-0094-6 Correspondence and reprints: Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.     

Prophase chromosome movements in living house cricket spermatocytes and their relationship to prometaphase,anaphase and granule movements

Chromosome and granule movements in meiotic prophase and prometaphase have been studied by time-lapse cinemicrography in live spermatocytes of the house cricket, Acheta domesticus. Chromosome movements in prophase cells, up to one hour or more before breakdown of the nuclear envelope, are described. These movements are frequent but saltatory; are based mostly at chromosome ends but also at kinetochores; occur in very intimate association with the inside of the nuclear envelope; are directed towards and away from the extranuclear centres (centrioles); tend weakly to accumulate bivalents round the two centres and reach a velocity of 0.65 m/sec. Saltatory movements in granules associated with extranuclear asters are remarkably similar in basic characteristics to the intranuclear chromosome movements. Surprisingly, the chromosome movements (and those of granules) are reversably blocked by colcemid (but not lumi-colcemid), and yet occur in the apparent absence of an intranuclear microtubule array. The movements cease at or shortly after breakdown of the nuclear envelope. However, kinetochore movements in very early prometaphase are similar in velocity and other respects to prophase movements; later prometaphase movements are clearly slower, and those of anaphase very much slower still. — The prophase movements suggest a two component model for motion: a non-microtubule, linear force producer together with microtubules with a skeletal, orientational role. Arguably, both these components are also necessary for chromosome movements in prometaphase and anaphase.This paper is dedicated to Dr. Sally Hughes-Schrader, whose beautiful work in mantids clearly presaged the existence of chromosome movements in late prophase of meiosis; and whose enthusiasm over chromosome movements in general it was my pleasure to share during my stay at Duke.     

Chromosome segregation in crane-fly spermatocytes: cold treatment and cold recovery induce anaphase lag

Anaphase lagging of autosomes was observed in 6.1±5.4% of the primary spermatocytes in untreated larvae of the crane fly, Nephrotoma suturalis. Lagging was induced by exposure of larvae to 6 ° C and during recovery at 22 ° C from exposure to 0.2, 2, and 6 ° C. The incidence of anaphase lag was maximal at 80 to 90 min of recovery. Induced lagging was observed at that recovery time after exposures of only 2.5 h to 2 or 0.2 ° C, but its incidence increased with longer exposures. As many as 85% of the cells in anaphase contained autosomal laggards after 61 h at 2 ° C and 80 to 90 min of recovery. At 2 ° C, cells reached the prophase-prometaphase transition, but spindles did not appear to form. Those cells proceeded through prometaphase during recovery, reaching mid-anaphase after 80 to 90 min of recovery. Chromosomes that lagged at anaphase during recovery from 2 ° C were observed in living cells to be half-bivalents derived from bivalents that congressed to the metaphase plate. One or both half-bivalents of any bivalent could lag. In some cells, one half-spindle had more half-bivalents than the other. Cells with autosomal laggards often did not cleave, and in uncleaved cells the second division employed spindles having two, three, or four poles. The basis of induced lagging might be a lapse in spindle attachment or motive force application at the start of anaphase or a failure of chromosomes to achieve proper orientation before the onset of anaphase.     

Biochemical analysis of actin in crane-fly gonial cells: evidence for actin in spermatocytes and spermatids--but not sperm

A biochemical assay employing DNase-I affinity chromatography, two- dimensional peptide analysis and SDS polyacrylamide gel electrophoresis was used to isolate, identify, and assess the amount of actin from gonial cells of the crane fly, Nephrotoma suturalis. Based on the analysis of cell homogenates under conditions in which all cellular actin is converted to the monomeric DNase-binding form, actin comprises approximately 1% of the total protein in homogenates of spermatocytes and spermatids. SDS gel analysis of mature sperm reveals no polypeptides with a molecular weight similar to that of actin. Under conditions that preserve native supramolecular states of actin, approximately 80% of the spermatocyte actin is in a sedimentable form whereas only approximately 30% of the spermatid actin is sedimentable. These differences could be meaningful with regard to strutural changes that occur during spermiogenesis. A comparative analysis of two- dimensional peptide maps of several radioiodinated actins reveals similarities among spermatocyte, spermatid, and human erythrocyte actins. The results suggest the general applicability of this approach to other cell types that contain limited amounts of actin.     

Backward chromosome movement in crane-fly spermatocytes after UV microbeam irradiation of the interzone and a kinetochore

Single anaphase chromosomes (in crane-fly spermatocytes) moved backwards after double irradiations with an ultraviolet light (UV) microbeam, first of the interzone and then of a kinetochore: the chromosome irradiated at the kinetochore moved backwards rapidly, across the equator and into the other half-spindle. High irradiation doses at the kinetochore were required to induce backward movement. Single irradiations of kinetochores or interzones were ineffective in inducing backward movements.     

Nucleotide requirements for anaphase chromosome movements in permeabilized mitotic cells: anaphase B but not anaphase A requires ATP

Permeabilized PtK1 cells continue to undergo anaphase chromosome movements provided MgATP is included in the lysis medium. However, chromosome-to-pole movement (anaphase A) and spindle elongation (anaphase B) differ with respect to nucleotide requirements. The rate of anaphase B depends on the concentration of ATP in the lysis medium; two-thirds the maximal rate is observed in 0.2 mM ATP. However, other nucleotides, such as ITP, CTP and GTP, cannot substitute for ATP. Spindle elongation is blocked by the addition of nonhydrolyzable ATP analogs. ADP, AMP and inhibitors such as vanadate, the magnesium chelator EDTA and sulfhydryl reagents. Anaphase does no require exogenous ATP and is unaffected by these inhibitors. These results are consistent with "dynein-like" ATPase involvement during spindle elongation, and rule out the possibility of tubulin-dynein and actomyosin mechanochemistry during anaphase A. I suggest that chromosome-to-pole movement involves the collapse of an elastic component in the spindle. Force generation could be provided by microtubule depolymerization or by the contraction of a nonmicrotubule microtrabecular lattice.     

Chromosome attachment to the spindle in crane-fly spermatocytes requires actin and is necessary to initiate the anaphase-onset checkpoint

Summary We investigated the possible involvement of actin in the attachment of chromosomes to spindles in crane-fly primary spermatocytes. In a previous study, cytochalasin D, an inhibitor of actin polymerisation, prevented bivalent attachment to microtubules when applied at prophase, but did not cause the detachment of already attached bivalents. We were able to detach the already attached bivalents by first treating prometaphase cells with an antitubulin drug, nocodazole, to disrupt spindle microtubules. 2 min after nocodazole addition, we added cytochalasin D, to disrupt actin filaments; then 2 min later nocodazole was removed, and the cells were kept in cytochalasin D until the time of normal anaphase. Double treatment with nocodazole and cytochalasin D blocked reattachment of bivalents to the spindle. Single treatment with nocodazole alone caused chromosome detachment but did not prevent reattachment when nocodazole was washed out. Extended treatment with cytochalasin D alone starting in prometaphase did not cause bivalents to detach from the spindle. These data suggest that actin is needed for attachment of bivalents to spindle microtubules. This protocol is relevant to the anaphase-onset checkpoint. From previous experiments it was argued that the anaphase-onset checkpoint recognises unattached chromosomes only after those chromosomes first interact with (become attached to) the spindle. Our experiments showed that anaphase disjunction occurred at normal times when bivalents were prevented from attaching to the spindle (by adding cytochalasin D in prophase), while anaphase disjunction was greatly delayed when previously attached bivalents were detached (with nocodazole) and then prevented from re-attaching (with cytochalasin D) in the double treated cells. Thus the anaphaseonset checkpoint recognises only those unattached bivalents that previously were attached to the spindle. Other results provided further indication that actin-microtubule interactions are important in spindle organisation. Nocodazole treatment for 4 min caused most microtubules to disappear: bivalents aggregated around remnant microtubules. When cytochalasin D treatment followed nocodazole treatment, remnant spindle microtubules were not seen, suggesting that actin interactions help stabilise those microtubules.Abbreviations CD cytochalasin D - NMBD nuclear-membrane breakdown - NOC nocodazole     

Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving. We examined actin and/or microtubules in irradiated cells by means of confocal fluorescence microscopy or serial-section reconstructions from electron microscopy. For each cell thus examined, chromosome movement had been recorded continuously until the moment of fixation. Kinetochore microtubules were completely severed by the ultraviolet microbeam in cells in which chromosomes continued to move poleward after the irradiation: none were seen in the irradiated regions. Similarly, actin filaments normally present in kinetochore fibres were severed by the ultraviolet microbeam irradiations: the irradiated regions contained no actin filaments and only local spots of non-filamentous actin. There was no difference in irradiated regions when the associated chromosomes continued to move versus when they stopped moving. Thus, one cannot explain motion with severed kinetochore microtubules in terms of either microtubules or actin-filaments bridging the irradiated region. The data seem to negate current models for anaphase chromosome movement and support a model in which poleward chromosome movement results from forces generated within the spindle matrix that propel kinetochore fibres or kinetochore stubs poleward.     

Centrifugation shearing exposes filamentous networks in cortical regions of crane-fly spermatocytes

Spermatocytes of the crane-fly, Nephrotoma suturalis, were attached to electron microscope grids and then sheared by applying centrifugal force. Transmission electron microscopy of exposed regions of the cell cortex revealed networks containing arrays of filamentous structures. Networks were present in sheared spermatocytes at all stages of meiosis. The networks of dividing spermatocytes (meta- through telophase) were denser and appeared to contain more aggregated material then networks of prophase cells. The appearance of networks in spermatocytes resembled actin-containing networks of sheared and detergent-extracted human erythrocytes. Networks treated with myosin subfragment 1 under conditions in which muscle F-actin was clearly decorated were not distinguishable from those of untreated cells. Exposure to deoxyribonuclease-1 caused the disruption of networks in sheared spermatocytes as well as in erythrocytes. The results of deoxyribonuclease experiments are interpreted as an indication that actin is a component of the cell cortex in crane-fly spermatocytes.     

Centromeric dots in crane-fly spermatocytes: meiotic maturation and malorientation

During the first meiotic division in crane-fly spermatocytes, the two homologs of a metaphase bivalent each bear two sister kinetochores oriented toward the same pole. We have previously reported treatments that increase the percentage of metaphase bivalents in which one or both homologs have bipolar malorientations: kinetochore microtubules] extending from a homolog toward both poles. The maloriented homologs lag at anaphase. Treatments that induce this behavior include: (a) recoverey from exposure to low temperatures or Colcemid or Nocodazole concentrations that prevent spindle formation but allow nuclear membrane breakdown, and (b) exposure to 6° C, a temperature that permits spindle assembly but slows progression through meiosis. Giemsa staining methods reveal two 0.5 m diameter dots at the centromeric region of each metaphase homolog; these often are more separated in maloriented homologs. This investigation was undertaken to assess whether this separation precedes the establishment of bipolar malorientation, and hence may be a cause of it, or is only a consequence of forces resulting from bipolar malorientation. Analysis showed that, in untreated cells, the average center-to-center distance between sister centromeric dots increases during the course of meiosis I. After the above-mentioned treatments, center-to-center distances similar to those normally seen in untreated half-bivalents at anaphase I were seen in bivalents, both after and before nuclear membrane breakdown. Longer exposure to temperatures that arrested meiosis increased the degree of dot separation. Based on our data, we conclude that normal orientation during the first meiotic division is aided by the close apposition of centromeric dots, and that a time-dependent maturation occurs causing centromeric dots to separate for the second meiotic division and facilitating orientation of sister kinetochores to opposite poles. If centromeric maturation occurs either prior to or during early stages of the first meiotic division, then it may contribute to persisting bipolar malorientation.     

Checkpoint control in crane-fly spermatocytes: unattached chromosomes induced by cytochalasin D or latrunculin treatment do not prevent or delay the start of anaphase

Summary Variable numbers of bivalents and sex chromosomes do not attach to the spindle when prophase or early prometaphase cranefly spermatocytes (2n=8) are treated with cytochalasin D or latrunculin. The unattached bivalents lie in the cytoplasm or at the spindle pole, and they do not delay onset of autosomal anaphase; sometimes they disjoin at the same time as the attached bivalents, so they respond to the global signals that initiate anaphase. Unattached sex chromosomes do not delay autosomal anaphase, either. Of various interpretations of these data, we think the best explanation is that the checkpoint system responds to physical rather than chemical cues; we think that the spindle is a tensegral structure, that chromosomes need to interact with the spindle in order to be recognised by the anaphase-onset checkpoint control, and that the physical interaction of chromosomes with spindle acts as a signalling network. Cytochalasin D and latrunculin treatments delay onset of sex chromosome anaphase (which normally occurs about 15 min after autosomal anaphase) and cause altered patterns of sex-chromosome segregation.     

Bivalent orientation and behavior in crane-fly spermatocytes recovering from cold exposure

At metaphase in crane-fly primary spermatocytes, the two sister kinetochores at the centromere of each homologue in a bivalent normally are adjacent and face the same pole; one homologue has all its kinetochore microtubules (kMTs) extending toward one pole and its partner has all its kMTs extending toward the opposite pole. In contrast, during recovery from exposure to 2 degrees C, one or both homologues in many metaphase bivalents had bipolar malorientations: all kMTs of one kinetochore extended toward one pole and some or all those of its sister extended toward the other. Metaphase sister kinetochores that had most of their kMTs extending toward the same pole were adjacent, and those with most extending toward opposite poles were separated from each other. Distances between homologous centromeres were similar to those in properly oriented bivalents. Maloriented bivalents were tilted relative to the spindle axis, and analysis of living cells showed that tilted configurations were rare during prometaphase in untreated cells but frequently arose in cold-recovering cells as initial configurations, then persisted through metaphase. This was in contrast to unipolar configurations of bivalents (configurations suggesting orientation of both homologous centromeres toward the same pole), which always reoriented shortly after the configuration arose. We conclude that in cold-recovering cells, bipolar malorientations are more stable than unipolar malorientations, and the orientation process is affected such that bipolar malorientations arise in bivalents upon initial interaction with the spindle and persist through metaphase.     

Distance segregation of sex chromosomes in crane-fly spermatocytes studied using laser microbeam irradiations

Univalent sex chromosomes in crane-fly spermatocytes have kinetochore spindle fibres to each spindle pole (amphitelic orientation) from metaphase throughout anaphase. The univalents segregate in anaphase only after the autosomes approach the poles. As each univalent moves in anaphase, one spindle fibre shortens and the other spindle fibre elongates. To test whether the directionality of force production is fixed at anaphase, that is, whether one spindle fibre can only elongate and the other only shorten, we cut univalents in half with a laser microbeam, to create two chromatids. In both sex-chromosome metaphase and sex-chromosome anaphase, the two chromatids that were formed moved to opposite poles (to the poles to which their fibre was attached) at speeds about the same as autosomes, much faster than the usual speeds of univalent movements. Since the chromatids moved to the pole to which they were attached, independent of the direction to which the univalent as a whole was moving, the spindle fibre that normally elongates in anaphase still is able to shorten and produce force towards the pole when allowed (or caused) to do so.     

Kinetochore microtubules in crane-fly spermatocytes: untreated, 2 degrees C-treated, and 6 degrees C-grown spindles

We investigated the involvement of kinetochore microtubules (kMTs) in mediating chromosome-to-pole connections in crane-fly (Nephrotoma suturalis and Nephrotoma ferruginea) spermatocytes. Two experimental treatments were used to yield spindles with reduced numbers of nonkinetochore microtubules (nkMTs). Short-term (10-15 min) exposure of spermatocytes to 2 degrees C caused depolymerization of the majority of nkMTs, resulting in a kMT:(kMT + nkMT) ratio of 0.76. Long-term (24h) exposure to 2 degrees C followed by recovery at 6 degrees C resulted in a kMT:(kMT + nkMT) ratio of 0.55, the spindle having more nkMTs than a 2 degrees C-treated spindle but fewer than an untreated spindle, in which the kMT:(kMT + nkMT) ratio was 0.27. The numbers and lengths of kMTs in 6 degrees C-grown spindles were similar to those in untreated cells, suggesting that the overall inhibition of MT assembly at 6 degrees C apparently did not affect the mechanism by which kMTs are formed. We observed most kMTs of early anaphase spindles to be long (greater than 3 microns), and many extended to the polar regions of the spindle. Thus, the crane-fly spindle appears not to be as atypical as it was previously suggested to be.