Jeremy David Pickett-Heaps 1940–2021^†

Green algae

Little was known, then, of the ultrastructure of algae, largely because their often highly vacuolated cells did not preserve well in the osmium-based fixatives used at the time; but Jeremy had the new fixation weapon of glutaraldehyde, which he would use to great effect on wheat seedlings. Stimulated by discussions with colleagues such as Terry O’Brien and Margaret McCully at the ANU’s Research School of Biological Sciences (to which Jeremy shifted his laboratory on acquiring a fellowship there in 1968), and along with his first post-doc, Larry Fowke, and first PhD candidate, Harvey Marchant, he trialled different concentrations and combinations of fixatives and buffers, and various dehydrating agents, embedding materials and stains to produce material that gave the best-possible representation of cells in life. He also quickly realised the benefits of always using either young, actively growing cultures or material collected directly from the wild: tenets that he emphasised to his students thereafter. As any electron microscopist knows, this is painstaking and often frustrating work, but when you get a ‘fix’ that is good, it is a joy to observe the thin-sections and discover as much as possible from the cellular detail that likely nobody has observed before.

With these well-preserved samples, Jeremy initially published a series of four papers on the fine structure of Chara, the most land-plant-like of the green algae, at all stages of growth and development.⁵ He then proceeded to investigate, with the members of his young research group, numerous and various other green algae—apparently, anything they could get their hands on. It soon became clear that there were clear delineations within the green algae with respect to the way in which they underwent cytokinesis. Jeremy’s key discovery was the identification of a mode of cytokinesis almost identical to that found in land plants, utilising the ‘phragmoplast’, which took place in only Chara and a few other green algae. Phragmoplasts were absent from the remaining large and diverse groups of green algae, such as Chlamydomonas (Chlamydomonas-like organisms were at the time considered to be the direct ancestors of the land plants). The phragmoplast is a precursor to the formation of a cell wall between daughter nuclei following mitosis: after anaphase, spindle microtubules in the phragmoplast hold the nuclei apart while also directing cytoplasmic vesicles into position for wall formation. The alternative mode of cytokinesis, which Jeremy and his laboratory found in all the other groups of green algae that they studied, involves the formation of a ‘phycoplast’. Jeremy coined this term⁶ to describe the process whereby the anaphase spindle breaks down and the daughter nuclei, adjacent to one another, become separated by new microtubules that form parallel to the plane of cytokinesis [This is different from phragmoplasts in which microtubules are always oriented perpendicular to the plane of cytokinesis]. The phycoplast functions not only to keep the daughter nuclei apart during division but, like the phragmoplast, plays a role in vesicle accumulation and wall formation. Jeremy recognised the phragmoplast as a fundamental feature of land plant development, and its presence in only a small group of green algae made those algae ancestral to the higher plants. Subsequent studies over the following decades, most recently from genetic sequencing data, have confirmed Jeremy’s initial findings as being pivotal in understanding the origins of land plants. His work on many major groups of green algae eventually led him to propose a different phylogeny for the Chlorophyta,⁷ and he continued to work on green algae even after retirement, producing many exceptional time-lapse videos of the structure, development and reproduction in green algae.

Diatoms

While continuing his interest in green algal evolution and ultrastructure, in the mid-1970s, Jeremy also turned his insight and curiosity to another group of algae, the diatoms. His specific interests centred around diatom cell division, the structures and processes involved in diatom cell wall morphogenesis, and around the mechanisms behind diatom motility. Members of his laboratory were constantly getting water samples from various ponds and creeks in the area to check for diatoms and other algae and brought samples even from far-away places that group members would go to on vacation; one pond in Boulder continues to be a source for diatom research to this day. Jeremy was always captivated by the early microscopy work of the botanist Robert Lauterborn and his exquisitely detailed drawings of the stages of algal development. In an effort to remind the community of often-forgotten great, past works, in 1984 he co-published a translation of Lauterborn’s 1896 treatise, demonstrating how modern optical and electron microscopy simply confirmed the excellent work of Lauterborn⁹ in understanding the nature of diatom mitosis.¹⁰

Jeremy was a master of the transmission electron microscope and the all-important methods that lie behind the generation of a final image of a preserved cell that best reflects the in vivo state. It was noted earlier how he developed techniques for observing the fine structure of green algae and so, too, with diatoms he was able to make significant advances. The hardened silica cell walls of diatoms makes the generation of thin sections of the cells for transmission electron microscopy difficult. Jeremy’s laboratory became expert at generating serial sections of fixed samples to reconstruct the internal cell organisation of diatoms many years before any type of 3D computer reconstruction was possible. This type of analysis became crucial in helping to understand the structure of diatom mitotic spindles (and mitotic spindles in general). In contrast to animal spindles, the highly ordered and localised spindles of diatoms, which are cylindrical and tightly packed, allowed Jeremy and graduate student, David Tippit, to follow the profiles of individual microtubules through an entire spindle by painstakingly comparing the micrographs of one section to those of the adjacent section. This work established that the spindle is in fact composed of two half spindles that interdigitate and overlap in the central region, and that the sliding apart of these interdigitated half-spindles accomplishes much of the pole-pole separation (anaphase B) that occurs during mitosis in diatoms.¹¹ The work also showed that the diatom centrosome, in contrast to that found in most animal cells, is not needed to maintain the organisation of mitotic spindles, but rather is involved in directing microtubules to help in the organisation and placement of cellular structures that form during cell division.¹²

Jeremy was keenly interested in how chromosomes accurately attach to the mitotic spindle, thus ensuring faithful separation of genetic material during mitosis. He considered proper attachment of the chromosomes to the spindle during prometaphase to be a seminal event in the evolution of all eukaryotic life. Once again, the diatom spindle helped to elucidate the basic functioning of the kinetochore during prometaphase when each chromosome pair attaches to the mitotic spindle. With the use of light and electron microscopy, it was determined that kinetochores function primarily by interacting with and sliding along microtubules from the poles. At prometaphase, one kinetochore of a chromosome pair moves toward either pole, while the second kinetochore is responsible for moving away from that pole, thus ensuring proper separation of the genetic material.¹³

Time-lapse recording

Jeremy operated with his usual enthusiasm to go beyond the static light or electron microscopic images and worked with the people in his group to record real-time and time-lapse images of numerous diatom processes. This included using 16 mm cine recordings to capture time-lapse well before video or digital time-lapse recording was easily available. By using perfusion chambers to introduce fixatives and drugs while making the live cell observations, then taking electron microscopic images of the fixed and treated cells, Jeremy and his co-workers were able to correlate the presence and localisation of specific cellular structures with dynamic processes. Such methods showed, for example, that a key role of the diatom microtubule organising centre (centrosome) was to help orchestrate the formation of the new cell walls following division.¹⁴ Drug treatments of developing diatoms also suggested the requirement for actin as well as microtubules in the localisation of the developing components of the diatom cell wall. Drug perfusion suggested that the sliding of half-spindles in anaphase B actively required energy input, while the movement of split chromosomes to poles (anaphase A) did not.¹⁵ Moreover, correlation of live video from high energy ultraviolet light ablation of sections of the diatom spindle, with subsequent electron microscopy by Roger Leslie in the laboratory, suggested that the two interdigitated half-spindles of mitosis were each composed of uniformly oriented microtubules, and that rates of microtubule depolymerisation were distinctly different at the two ends of microtubules.¹⁶

Being acutely interested in cell dynamics, Jeremy also had the opportunity to work with Lesley Edgar, a visiting post-doc in his group, to investigate the unresolved problem of gliding motility in diatoms. Using a combination of live microscopy and electron microscopy of the underlying cell ultrastructure, the Edgar/Pickett-Heaps model for motility was developed, in which underlying actin cables provide structure over which trans-cellular components (using myosin) can move down the raphe fissure (a slit in the cell wall of many benthic diatoms) to provide movement.¹⁷ While there are still details that remain unresolved, more than forty years later this model continues to be the major working hypothesis for understanding the cellular movement of diatoms with raphes.

As the 1980s progressed, Jeremy found that procuring funding was becoming increasingly difficult in the US, particularly for those cell structure and dynamics labs like his that were not focused on the molecular and genetic aspects of cells. The increasing pressure to obtain external funding sources to continue his work, and his desire to return home to Australia, led to him taking a position at the University of Melbourne in 1988.

Animal mitosis

Another visitor to the laboratory in the early 1990s was this Memoir’s second author, Arthur (Art) Forer (Fig. 4) and this led to a collaboration studying animal cell mitosis that was to last some fifteen years. Jeremy’s doctoral studies on cell division and preprophase bands naturally led him to become interested in the workings of mitosis, the separation of chromosomes via the mitotic spindle that happens before division of the cell. At that time, electron microscope studies of mitotic spindles showed that microtubules extend between each chromosome and the spindle pole towards which the chromosome moves; most observers considered that these ‘kinetochore’ microtubules produced the forces that moved the chromosomes poleward. Jeremy was no exception: he studied spindle microtubules in dividing diatoms and other algae, trying to understand how they produced force. Experiments with Tim Spurck (a research assistant who later became his graduate student) in the early 1980s,¹⁹ on the diatom Hantzschia, told him that kinetochore microtubules did not produce force but instead acted as guides for the direction in which the chromosomes should move and that the forces that moved the chromosomes were rather produced by a ‘spindle matrix’. In normal Hantzschia cells, kinetochore microtubules are embedded in electron-dense material. He and Spurck depolymerised kinetochore microtubules with colchicine. Spindle structure was maintained when the kinetochore microtubules were depolymerised because the two spindle poles were connected by a separate bundle of microtubules that were not depolymerised by colchicine. When the kinetochore microtubules disappeared, each chromosome moved randomly to one of the two poles (illustrated in Fig. 5). Jeremy and Spurck concluded that something other than microtubules propelled the chromosomes to the poles, a spindle matrix in the form of the electron dense material associated with the kinetochore microtubules. They concluded that microtubules act to guide chromosomes to move in the proper direction and also act as ‘governors’ of speed (since the rate of chromosome movement in untreated cells is governed by the rate of microtubule depolymerisation). This key experiment on diatom mitosis was a basis for Jeremy’s thinking about mitotic mechanisms as he began experiments on animal cells.

Fig. 4.

After lunch at the Danish Club in Melbourne, ~1995. From L-R: Tim Spurck, Sandra Forer, Art Forer, Jeremy Pickett-Heaps, Julianne Pickett-Heaps. Photographer unknown.

Fig. 5.

The four electron micrographs are illustrations of mitotic cells before colchicine addition (Image a) and after colchicine addition (Image b); the two images at the bottom are at higher magnification, illustrating the kinetochores at the poles after removal of their connecting microtubules. The kinetochore arrangements at the poles allowed Pickett-Heaps and Spurck to deduce what happened, as illustrated in the coloured cartoons of control and colchicine-treated cells (Pickett-Heaps and Spurck 1982a). In control cells, each chromosome at metaphase is composed of two chromatids that at anaphase split into two chromatids that then move to opposite poles. After colchicine treatment, on the other hand, the chromosomes did not split into two but each one, consisting of two chromatids, moved to one of the two poles at random (Adapted from Pickett-Heaps and Forer 2009).

Jeremy began to work with mitotic animal cells around 1985, perhaps to make his interpretations derived from Hantzschia and other ‘odd’, ‘unusual’, non-canonical cells more relevant to colleagues who considered that mitosis occurs only as seen in ‘standard’ animal cells. His early experiments used tissue-cultured marsupial (PtK) cells;²⁰ later he studied cultured newt lung cells. The kinetochore microtubules in these spindles are not embedded in electron-dense material (that is, there is no visible ‘matrix’) but experiments using an focused ultraviolet light to damage kinetochore microtubules confirmed that a spindle matrix functions to move chromosomes in animal cells just as in diatoms. The experiments were these.

Jeremy and colleagues monitored the presence of spindle microtubules in living cells using polarisation microscopy. When a slit-shaped beam of ultraviolet light irradiated across the newt or PtK-cell spindle between the chromosomes and a pole, the birefringence (microtubules) disappeared in the irradiated region, severing the spindle into two pieces—a polar region with microtubules extending toward the equator but ending blindly in the irradiated region, and the rest of the spindle not attached the severed part. The rest of the spindle seemed ‘normal’: severed microtubules extended in one direction from the chromosomes, towards one pole, and ended blindly in the irradiated region while the non-severed microtubules extended to the pole in the other direction and connected the chromosomes to the other pole. Shortly after the microtubules were severed, the ‘amputated’ pole moved towards the chromosomes at the equator. Since this movement occurred in the absence of microtubule connections across the irradiated region, the movements were not caused by microtubules but rather forces for movement were produced by an underlying spindle ‘matrix’. In the article describing these results,²¹ however, Jeremy and colleagues suggested that the movements of the poles to the equator arose from growth of additional microtubules from the pole; they did not discuss the possibility that movement arose from a spindle matrix. Forer worked in Jeremy’s laboratory in 1993, as a spindle matrix skeptic, and the day he entered Jeremy’s laboratory was challenged by Jeremy with: ‘what causes the pole to move toward the equator when the connecting microtubules are cut?’ Jeremy argued that there must be something underlying the microtubules (a spindle matrix) pulling the pole toward the equator. Challenging Forer forcefully in this way, several times, meant that regardless of what Jeremy wrote in the articles, he personally found this experiment convincing of spindle matrix activity. He did not convince Forer at that time that a spindle matrix produced forces to move chromosomes in animal cells, but he did so later.

One key result arose from using focused ultraviolet light to sever individual kinetochore fibre microtubules during anaphase. Severing kinetochore microtubules should stop the associated chromosomes from moving if the microtubules produce the force, but they did not stop. In newt lung cells, for example, not only did the associated chromosome not stop moving, it accelerated.²² This is exactly what one would expect if an invisible matrix pushed the chromosome poleward: because severing microtubules removes the need to depolymerise them in order for chromosomes to move, cutting the microtubules removes the speed limitation so the movements should accelerate.²³ These and related experiments convinced Forer that there were force producers (a spindle matrix) underlying the spindle microtubules. Alas, most colleagues still are not convinced that a spindle matrix or anything other than spindle microtubules produce force.

Some colleagues have accepted that a spindle matrix underlies the microtubule spindle structure, but they say its role is in transporting essential regulatory components;²⁴ only a few considered it to have a possible role in chromosome movement.²⁵ To look for additional evidence, Jeremy and colleagues studied non-microtubule spindle components that might produce forces. The two major motile force producers for intracellular movements are (a) microtubules and their associated motor proteins, and (b) actin and its motor protein myosin. If not microtubules, why not actin? Jeremy and colleagues had found actin in various spindles, including in Oedogonium,²⁶ and they used actin inhibitors to alter mitotic processes.²⁷ From these and other data Jeremy argued that spindles incorporated actin and myosin when they evolved from primitive motile systems.²⁸ Our colleagues by and large remain unconvinced that actin is involved.

Red algae

In the mid-1990s, Jeremy began another important collaboration, with John West, an emeritus professor of phycology who, at that time, had recently moved to Melbourne’s School of Botany from UC, Berkeley. John brought to Melbourne a large collection of red algal cultures and Jeremy immediately pounced on this treasure trove and began filming various unicells and the reproductive spores from multicellular species. Red algae were assumed to have no motile stages because members of the phylum universally lacked flagella. There were a few unsupported claims of motility in the early literature, but it wasn’t until Jeremy filmed the activity of these cells while keeping them alive for many hours in a perfusion chamber that these reports could be confirmed. Of the more than twenty five species that Jeremy filmed, most turned out to have motile stages. These findings were significant because, at the time, phycologists had little to no understanding of red algal motility and the movements shown in his recordings were potentially critical to the survival of species by enabling optimisation of sites for spore germination. As surprising as these findings were, Jeremy’s microcinematography of sexual reproduction in red algae was truly amazing: from spermatial release to nuclear migration and fertilisation, he again observed and beautifully recorded dynamic cellular events that were previously unknown.²⁹

Microcinematography

Jeremy was a great proponent and practitioner of observing living cells, not least to validate what he saw in static electron micrographs. His progression into the world of time-lapse imaging, first using analogue and later using digital microcinematography, can be seen as a complement to his many achievements in electron microscopy. His reasoning for this progression to live imaging may be best summarised by this quote from the introduction to his commercial website Cytographics,³⁰ which he started in the mid 1990s with his wife, Julianne:

As a young scientist, I became fascinated by electron microscopy. In the late sixties, this technique was dramatically transforming our concept of cellular structure. I concentrated upon cell division and morphogenesis in green algae and then diatoms. As this work progressed, I became increasingly frustrated at trying to recreate dynamic cellular events solely from static images. A turning point in my career came when I first saw the extraordinary sight of a live diatom undergoing mitosis at high magnification. After borrowing a 16 mm time-lapse camera, I was soon filming algae doing all the things I had studied with the electron microscope. Since then, I have built up a laboratory devoted to the high-resolution video imaging and recording of all sorts of cells and microscopic organisms going about their complex and extraordinary lives.

This transition to live cell imaging might seem easy when compared to the many innovative techniques he successfully developed for the preparation of plant and algal cells for transmission electron microscopy. But similar innovations were required to prepare living cells for surviving and performing naturally during long periods of time-lapse filming under bright lights, particularly the processes of cell division and morphogenesis for which his resulting films and videos are famous. To this end, Jeremy and Tim Spurck developed the aforementioned micro-chambers to facilitate the high-resolution filming of live cells, including chambers with a continuous flow of media to replenish the cells. Having successfully achieved that, they formed collaborations with a number of local electronic experts to develop a shutter system that acted in concert with the time-lapse apparatus and camera so that cells were only subjected to the light (and resulting heat) for the limited time required for a single frame to be exposed. The only limitation was the need to refocus over long periods of time, as even the best microscopes gradually fade out of focus with time. His students and colleagues remember his mad dashes back to the laboratory after dinner, and into the night, to refocus the microscope during time-lapsed sequences that could last up to 18 h or more, also replenishing the media bottle supplying nourishment to the cells being filmed. Like all of Jeremy’s endeavours, he sought perfection, and he endlessly worked towards finding better ways to keep cells alive and functioning normally, as well as improving the technology for time-lapse microscopy.

In addition to his scientific discoveries published in a range of cell biological and phycological journals, Jeremy was equally interested in inspiring a much broader audience with the wonders and diversity of cells. It was with this impetus that Cytographics was launched. A series of DVDs were produced that have pleased teachers, students and naturalists the world over. DVDs produced include those designed for introductory biology courses, such as Living Cells: Structure, Function and Diversity; for specialists taking courses in phycology or protistology, such as Diatoms: Life in a Glass House and Reproduction in the Red Algae; or cell biology titles such as The Dynamics and Mechanics of Mitosis.

Later in his career, Jeremy collaborated with a former student and friend, Drew Berry (now lead animator at the Walter and Eliza Hall Institute’s WEHI-TV), to combine living-cell sequences with dazzling animations. The link to an edited selection of the many movies made by Jeremy, Tim and Drew is included in Supplementary material.

Many of Jeremy’s long-held and sometimes prescient thoughts around the state of cell biology are documented in the Introduction to Green Algae.³¹ He despised ‘the dominance of animal and other highly evolved cells in formulating theories and dogmas in cell biology… it is an unbalanced approach, quite dangerous and beset with pitfalls’. A good example is that centrioles, at that time (and, in some quarters, still), were considered by text books to be an essential organiser of the mitotic spindle, yet ‘discerning students and teachers, who perforce must swallow these dogmas, cannot help being mystified by the ability of plant cells to divide perfectly well without centrioles’. He championed the algae as organisms complementary to the animals and plants for studies of cell biology: ‘I believe that algae in general represent a fascinating and vast range of organisms whose potential for the study of fundamental processes of life has been largely unexploited …’, and, ‘this book … hopefully supports my contention that these organisms should command more interest from biologists because they provide excellent material for the deeper investigation of diverse biological phenomena.’

Jeremy’s early work was readily accepted by the field; his later work on spindle matrix and actin was not. Part of the reason may be that much of the work involved ‘crazy’ ideas derived from studies of diatoms and crane-fly spermatocytes, both cell types with non-canonical mitoses, in a world where much of the important research on stereotypical animal cells is directed at medical applications. Also, colleagues may not appreciate how good an observer Jeremy was: one may not understand his conclusions if one is not used to scrutinising cell and organelle behaviour as Jeremy did. Nor are many willing to change direction to consider alternate interpretations when confronted with contrary data from the cell, as Jeremy did.