Cell division inCarteria crucifera (chlorophyta): The role of the endomembrane system and phycoplast

Summary Phycoplast-mediated cytokinesis in the primitive green algal flagellate,Carteria crucifera, has been examined by electron microscopy. The key developmental foci during cell division are mobile centriole-MTOCs which control mitotic spindle formation, the establishment of the plane of cytokinesis, the initiation of the cytokinetic furrow, the formation of the phycoplast and the formation of morphogenetic microtubular arrays. The cytokinetic cleavage mechanism entails an ingressive furrowing closely associated with a prolific network of internuclear endoplasmic reticulum. Dictyosome activity is limited to the cleavage initiation zone and is responsible for the production of wall precursor-containing vesicles. Dictyosome materials do not contribute directly to the growing furrow edge. Potassium antimonate staining patterns reveal the cytokinetic ER as a storage/control site for calcium during cytokinesis. Discussion of possible models concerning this cytokinetic mechanism is presented.     

FINE STRUCTURE OF MITOSIS AND CLEAVAGE IN FRIEDMANNIA ISRAELENSIS (CHLOROPHYCEAE: CHLOROSARCINACEAE)1

Observations on the ultrastructure of Friedmannia israelensis Chantanachat & Bold revealed the presence of a phycoplast and zoospores with cruciate rootlets. During mitosis, the nuclear envelope partially disintegrates and the basal bodies remain at the cell surface on either side of the developing cleavage furrow. The events during mitosis and cleavage in Friedmannia resemble those reported in the other green algae, Platymonas and Pleurastrum.     

COLONY DEVELOPMENT IN EUDORINA ELEGANS (CHLOROPHYTA,VOLVOCALES)1

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.     

Coordination of division events in theChlamydomonas cell cycle

Summary At concentrations that did not affect growth, hydroxyurea and 2¹-deoxyadenosine inhibited DNA synthesis inChlamydomonas. Evidence that initiation of mitosis is dependent upon completion of DNA replication was provided by the arrest of inhibited cells with undivided nuclei containing undispersed nucleoli. Initiation of cytokinesis is not dependent upon progress of nuclear division since, in arrested cells, cleavage microtubules became deployed in a phycoplast and a cleavage furrow developed fully, until obstructed by the undivided nucleus. Chloroplast constriction and division also continued independently of nuclear division. It is concluded that nuclear division, cytoplasmic cleavage and chloroplast division are in separate sequences of dependent events. This is supported by flexibility of their relative timing in successive divisions, since after the first commitment to divide nuclear division is followed by initiation of cleavage and then chloroplast division, whereas following subsequent commitments these events occur in reverse time order. This flexibility of order indicates changing rates of progress through separate sequences of events.Deposition of wall material was dependent upon the completion of cytokinesis, but this inhibition of wall deposition by incomplete cytokinesis did not extend to other daughters within the same mother cell.These observations are correlated with our earlier data concerning the rate-limiting control points for division and a model for the coordination of division events is presented. The relationships between different plant cell cycles is discussed in view of the findings presented.     

Diatom motility: An explanation and a problem

Except for the lack of a centriole, interphase cell morphology and cell division in Stichococcus is similar to that in Klebsormidium. The cell in Stichococcus is largely filled by a chloroplast and pyrenoid, at the side of which are two mitochondria and one small peroxisome. The chloroplast/pyrenoid cleaves early in prophase, probably completely, and the nucleus is inserted between the two halves. A band of 3–5 microtubules always encircles the prophase nucleus; these disappear by metaphase. The spindle is open, the daughter nuclei remain far apart at telophase and during cytokinesis, and vacuoles collect between them; no phycoplast is associated with the cleavage furrow.

These results indicate a close phyletic relationship between Stichococcus and Klebsormidium, two organisms which are now considered to be more closely related to the progenitors of the higher land plants than most of the other members of the Ulotrichales.     

CELL DIVISION IN THE FILAMENTOUS PLEURASTRUM AND ITS COMPARISON WITH THE UNICELLULAR PLATYMONAS (CHLOROPHYCEAE)1

At prophase in Pleurastrum, extranuclear spindle microtubules develop from the region of centrioles, which lie lateral to the nucleus midway between the future sites of the metaphase spindle poles. The microtubules then move laterally to overarch the nucleus and finally become incorporated into the spindle. The centrioles do not migrate and therefore lie in the same plane as the chromosomes at metaphase. At telophase, 2, more different systems of microtubules develop from the vicinity of the centrioles—a phycoplast and extensive arrays of microtubules that ensheath the daughter nuclei. Cell division in the filamentous Pleurastrum is compared to that in the green flagellate, Platymonas. The similarities between cell division in the 2 algae are interpreted as evidence: (i) that rhizoplasts (which in Platymonas resemble myofibrils) are somehow homologous to microtubules; and, (ii) that cell division in Pleurastrum differs from cell division in other examined filamentous chlorophycean genera because Pleurastrum has an independent evolutionary origin from a monad with Platymonas-like characteristics.     

TWO TYPES OF CYTOPLASMIC CLEAVAGE IN THE COENOCYTIC SOIL ALGA PROTOSIPHON BOTRYOIDES (CHLOROPHYCEAE)1

Cytokinesis in the coenocytic green alga Protosiphon botryoides (Kütz.) Klebs was studied with transmission electron microscopy. In vegetative cells, nuclei with associated basal bodies and dictyosomes are scattered throughout the cytoplasm. Mature cells may develop either multinucleate resting spores (coenocysts) or uninucleate zoospores. Cytokinesis may be preceded by contraction of the protoplast due to the disintegration of vacuoles that are present in larger, siphonous cells. The formation of coenocysts in ageing, siphonous cells, is signalled by cleavage of the chloroplast and the development of arrays of phycoplast microtubules in one or more transversely oriented planes through the cell. Nuclei with associated basal apparatuses stay dispersed throughout the cytoplasm; the basal bodies apparently are not involved in organization of the phycoplast. The plasma membrane invaginates, resulting in a centripetal cleavage of the protoplast into two or more multinucleate daughter protoplasts. Simultaneously, wall material is deposited along the outside of the daughter protoplasts by dictyosome-derived vesicles, and finally two or more thick-walled coenocysts are formed. The formation of zoospores, on the other hand, is signalled by clustering of the nuclei in one or more groups depending on the shape of the mother cell. The nuclei become arranged with the associated basal apparatuses facing toward the center of the cluster. Bundles of phycoplast microtubules develop between the nuclei, radiating from the center of a cluster toward the plasma membrane; basal apparatuses or associated structures apparently are involved in organization of the phycoplast. Cleavage furrows grow out centrifugally along these bundles of micro-tubules, fed by dictyosome-derived vesicles. No wall material is deposited. An additional mitotic division occurs during cleavage, and finally numerous uninucleate, wall-less, biflagellate zoospores are formed. The ultrastructural features of the two different types of cytoplasmic cleavage associated with two different types of daughter cells have not previously been reported for chlorophycean algae.     

CELL DIVISION IN BULBOCHAETE. I. DIVISIONS UTILIZING THE WALL RING1

Cell division in Bulbochaete closely resembles that of Oedogonium, particularly in the involvement of a ring in cell elongation, the structure of the spindle, the existence of complex kinetochores, and the method of cross-wall formation using a phycoplast. Some minor differences between the 2 genera are found. In contrast to Oedogonium, the filaments of Bulbochaete are branched. The site and direction of branching are initially determined by a subtle change in the morphology of the wall, which invariably (if the cell divides) leads to the asymmetrc division that forms a hair cell (these events will be described separately). The position of the wall ring is always precisely determined as in Oedogonium, by the position of a very characteristic weakening in the wall; once a hair cell has been formed, this weakening is located underneath the hair, and all subsequent division and elongation in the cell subtending the hair will necessarily be in the direction of that hair (ie, thereby forming and increasing the length of a branch).     

Arv1 promotes cell division by recruiting IQGAP1 and myosin to the cleavage furrow

Cell division is strictly regulated by a diversity of proteins and lipids to ensure proper duplication and segregation of genetic material and organelles. Here we report a novel role of the putative lipid transporter ACAT-related protein required for viability 1 (Arv1) during telophase. We observed that the subcellular localization of Arv1 changes according to cell cycle progression and that Arv1 is recruited to the cleavage furrow in early telophase by epithelial protein lost in neoplasm (EPLIN). At the cleavage furrow Arv1 recruits myosin heavy chain 9 (MYH9) and myosin light chain 9 (MYL9) by interacting with IQ-motif-containing GTPase-activating protein (IQGAP1). Consequently the lack of Arv1 delayed telophase-progression, and a strongly increased incidence of furrow regression and formation of multinuclear cells was observed both in human cells in culture and in follicle epithelial cells of egg chambers of Drosophila melanogaster in vivo. Interestingly, the cholesterol-status at the cleavage furrow did not affect the recruitment of either IQGAP1, MYH9 or MYL. These results identify a novel function for Arv1 in regulation of cell division through promotion of the contractile actomyosin ring, which is independent of its lipid transporter activity.     

The position of cleavage furrow in cultured <Emphasis Type="Italic">L-929</Emphasis> and <Emphasis Type="Italic">CHO</Emphasis> cells

The position of the cleavage furrow (random or otherwise) was studied on cultured L-929 (NCTC, clone 929) and CHO cells. CHO cells were seeded uniformly on the surface of Petri dishes; L-929 cells were grown as colonies so that migrating cells could be watched. Cell behavior was registered by time-lapse imaging. Two parameters were analyzed on captured images: the angle between the cell polarization axis and cleavage furrow and the angle between the cell polarization axis or cleavage furrow and the horizontal axis of the image field. It was shown that the position of CHO cells in the dish plane and the value of the angle between the cell polarization axis and the cleavage furrow were random. The L-929 cells migrating from the colony were orientated such that their polarization axis was directed to the colony center and the cleavage furrow was perpendicular to this axis. The nonrandom position of cultivated cells during mitosis and their cleavage furrow during the telophase are discussed.     

CELL DIVISION IN CHLAMYDOMONAS MOEWUSII1

Cell division in Chlamydomonas moewusii is described. The cells become immobile with flagellar abscission prior to mitosis. The basal bodies migrate toward the nucleus and become intimately associated with the nuclear membrane which is devoid, of ribosomes where adjacent to the basal bodies. The basal bodies replicate at preprophase. The nucleolus fragments at this stage. By prophase the basal body pairs have migrated, to the nuclear poles. Spindle fibers become prominent in the nucleus. The nuclear membrane does not fragment. The nucleus assumes a crescent-form by metaphase. Polar fenestrae are absent. Kinetochores appear at anaphase. An interzonal spindle elongates as the chromosomes move to the nuclear poles. Daughter nuclei become abscised by an ingrowth of nuclear membrane, leaving behind a separated, degenerating interzonal spindle. Ribosomes reappear on the outer nuclear membrane at late telophase. Nucleoli reform early in cytokinesis. The cleavage furrow, associated microtubules, and endoplasmic reticulum comprise the phycoplast. Cytokinesis proceeds rapidly after the completion of telophase. The basal body-nucleus relationship becomes reorganized into the typical interphase condition late in cytokinesis. Specific and predictable organelle rearrangements during mitosis have been described. Cell division in C. moewusii is compared with other algae, especially C. reinhardi.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

FINE STRUCTURE OF CELL DIVISION IN CHLAMYDOMONAS REINHARDI : Basal Bodies and Microtubules

Cell division in log-phase cultures of the unicellular, biflagellate alga, Chlamydomonas reinhardi, has been studied with the electron microscope. The two basal bodies of the cell replicate prior to cytokinesis; stages in basal body formation are presented. At the time of cell division, the original basal bodies detach from the flagella, and the four basal bodies appear to be involved in the orientation of the plane of the cleavage furrow. Four sets of microtubules participate in cell division. Spindle microtubules are involved in a mitosis that is marked by the presence of an intact nuclear envelope. A band of microtubules arcs over the mitotic nucleus, indicating the future cleavage plane. A third set of microtubules appears between the daughter nuclei at telophase, and microtubules comprising the "cleavage apparatus" radiate from the basal bodies and extend along both sides of the cleavage furrow during cytokinesis. Features of cell division in C. reinhardi are discussed and related to cell division in other organisms. It is proposed that microtubules participate in the formation of the cleavage furrow in C. reinhardi.     

Molecular and cellular dynamics of early embryonic cell divisions in Volvox carteri

Cell division is fundamental to all organisms and the green alga used here exhibits both key animal and plant functions. Specifically, we analyzed the molecular and cellular dynamics of early embryonic divisions of the multicellular green alga Volvox carteri (Chlamydomonadales). Relevant proteins related to mitosis and cytokinesis were identified in silico, the corresponding genes were cloned, fused to yfp, and stably expressed in Volvox, and the tagged proteins were studied by live-cell imaging. We reveal rearrangements of the microtubule cytoskeleton during centrosome separation, spindle formation, establishment of the phycoplast, and generation of previously unknown structures. The centrosomes participate in initiation of spindle formation and determination of spindle orientation. Although the nuclear envelope does not break down during early mitosis, intermixing of cytoplasm and nucleoplasm results in loss of nuclear identity. Finally, we present a model for mitosis in Volvox. Our study reveals enormous dynamics, clarifies spatio-temporal relationships of subcellular structures, and provides insight into the evolution of cell division.

Analysis of cell divisions of the microalga Volvox reveals enormous dynamics of cytoskeletal and membranous structures with coordination of intranuclear spindle formation by cytosolic centrosomes.

IN A NUTSHELLBackground: Mitosis, a type of cell division, is fundamental to all eukaryotic life and must be carried out very accurately. Even though the process of mitosis itself is highly conserved among eukaryotes, there are significant differences between animals, fungi, plants, and algae. From an evolutionary point of view, the green alga Volvox carteri used here possesses both key animal and plant functions and it exhibits important features of the last common eukaryotic ancestor that have been lost in other lineages. Prior to our work, a comprehensive in vivo analysis of the entire process of cell division in green algae was lacking.Question: How exactly does cell division work in green algae? How do the cytosolic centrosomes deal with the persistent nuclear envelope in this process? What is the relationship between different microtubular structures?Findings: Our study reveals enormous dynamics during mitosis, clarifies spatio-temporal relationships of subcellular structures, and provides insights into evolution of cell division. Although the nuclear envelope does not break down during early mitosis of Volvox, it becomes permeable and the nucleus temporarily loses its identity. Two microtubule-organizing centers, the centrosomes, located immediately outside the nuclear envelope participate in initiation of the mitotic spindle formation inside the nuclear envelope. This process also defines the orientation of the mitotic spindle. In cytokinesis, an algae-specific microtubule structure, the phycoplast, replaces the spindle. The microtubules of the phycoplast may play a direct role in promoting the cell membrane invagination of the cleavage furrow.Next steps: How are the massive rearrangements of subcellular structures regulated? What happens at the nuclear pores when the nuclear envelope becomes permeable at the onset of mitosis? What determines in later embryogenesis which cells then divide asymmetrically rather than symmetrically?     

The dynamic surface of dividing cyanelles and ultrastructure of the region directly below the surface in <Emphasis Type="Italic">Cyanophora paradoxa</Emphasis>

The cyanelles of glaucocystophytes are probably the most primitive of known extant plastids and the closest to cyanobacteria. Their kidney shape and FtsZ arc during the early stage of division define cyanelle division. In order to deepen and expand earlier results (Planta 227:177–187, 2007), cells of Cyanophora paradoxa were fixed with two different chemical and two different freeze-fixation methods. In addition, cyanelles from C. paradoxa were isolated to observe the surface structure of dividing cyanelles using field emission scanning electron microscopy (FE-SEM). A shallow furrow started on one side of the division plane. The furrow subsequently extended, covering the entire division circle, and then invaginated deeply, becoming clearly visible. The typical FtsZ arc was 2.3–3.4 μm long. This length matches that of the cleavage furrow observed using FE-SEM. The cyanelle cleavage furrows are from one-fourth to one-half of the circumference of the division plane. The shallow furrow that appears on the cyanelle outer surface effectively changes the division plane. Using freeze-fixation methods, the electron-dense stroma and peptidoglycan could be distinguished. In addition, an electron-dense belt structure (the cyanelle ring) was observed inside the leading edge at the cyanelle division plane. The FtsZ arc is located at the division plane ahead of the cyanelle ring. Immunogold-TEM localization shows that FtsZ is located interiorly of the cyanelle ring. The lack of an outer PD ring, together with the arch-shaped furrow, suggests that the mechanical force of the initial (arch shaped) septum furrow constriction comes from inside the cyanelle.     

MITOSIS IN THE OCTAFLAGELLATE PRASINOPHYTE,PYRAMIMONAS AMYLIFERA (CHLOROPHYTA)1

Cell division is described in the octaflagellate prasinophyte Pyramimonas amylifera Conrad and is compared in related genera. Basal bodies replicate at preprophase and move toward the poles. Cells remain motile throughout division. The nuclear envelope disperses and chromosomes begin to condense at prophase. Pairs of multilayered kinetochores are evident on the chromosomes of the metaphase plate. Spindle microtubules extending from the region of the basal bodies and rhizoplasts attach to the kinetochores or extend from pole to pole. Numerous vesicles and ribosomes have entered the nuclear region and the incipient cleavage furrow invaginates. The chromosomes move toward the poles at anaphase leaving a broad interzonal spindle between the two chromosomal plates. The nuclear envelope reforms first around the chromatin on the side adjacent to the spindle poles and later on the interzonal side. The cleavage furrow progresses into the interzonal spindle at telophase. By late telophase the nucleoli have reformed and the chromosomes have decondensed. The interzonal spindle has not been observed late in telophase. As the cleavage furrow nears completion the cells begin to twist and contort, ultimately separating the two cells.     

Ultrastructure of vegetative organization and cell division in the freshwater red algaCompsopogon

Summary Uniseriate filaments of the freshwater red algaCompsopogon coeruleus were examined by transmission electron microscopy for details of vegetative organization and cell division with the goal of providing useful taxonomic characters. Each cell's single, complex chloroplast contains a peripheral encircling thylakoid, and unlike the vast majority of red algae, the cis-regions of dictyosomes are not consistently juxtaposed with mitochondria. These subcellular features, which are present in all examined genera in theCompsopogonales, Erythropeltidales, andRhodochaetales, along with certain unique reproductive characteristics, unify these three orders. During mitosis in uncorticated axial cells, a small, ring-shaped nucleus associated organelle (NAO) is located at each division pole, an intranuclear spindle comes to a moderately acute focus at the flattened, fenestrated metaphase-anaphase division poles and perinuclear ER partially encloses dividing nuclei, including a well-developed interzonal midpiece. The cleavage furrow penetrates the large, central vacuolar region to separate daughter nuclei. These cell division features most closely resemble the pattern described for the orderCeramiales. Our observations of vegetative and dividing cells ofC. coeruleus supplement the growing volume of evidence in favour of uniting all red algae into a single class without subclass designations.Abbreviations ER endoplasmic reticulum - IZM interzonal midpiece - MT microtubule - MTOC microtubule organizing center - NAO nucleus associated organelle - NE nuclear envelope - PER perinuclear endoplasmic reticulum     

Polo-like kinase 1 directs the AMPK-mediated activation of myosin regulatory light chain at the cytokinetic cleavage furrow independently of energy balance

It has been recently proposed that AMP-activated protein kinase (AMPK) might indirectly promote the phosphorylation of MRLC (myosin II regulatory light chain) at Ser19 to regulate the transition from metaphase to anaphase and the completion of cytokinesis. Although these findings provide biochemical support for our earlier observations showing that the active form of the α catalytic AMPK subunit associates dynamically with essential mitotic regulators, several important issues remained unexplored. Does glucose starvation alter the ability of AMPK to bind to the mitotic apparatus and travel from centrosomes to the spindle midzone during mitosis and cytokinesis? Does AMPK activate MRLC exclusively at the cleavage furrow during cytokinesis? What is the mitosis-specific stimulus that activates the mito-cytokinetic AMPK/MRLC axis regardless of energy deprivation? First, we confirm that exogenous glucose deprivation fails to alter the previously described distribution of phospho-AMPKα^Thr172 in all of the mitotic phases and does not disrupt its apparent association with the mitotic spindle and other structures involved in cell division. Second, we establish for the first time that phospho-AMPKα^Thr172 colocalizes exclusively with Ser19-phosphorylated MRLC at the cleavage furrow of dividing cells, a previously unvisualized interaction between phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 that occurs in cleavage furrows, intercellular bridges and the midbody during cell division that appears to occur irrespective of glucose availability. Third, we reveal for the first time that the inhibition of AMPK mitotic activity in response to PLK1 inhibition completely prevents the co-localization of phospho-AMPKα^Thr172 and phospho-MRLC^Ser19 during the final stages of cytokinesis and midbody ring formation. Because PLK1 inhibition efficiently suppresses the AMPK-mediated activation of MRLC at the cytokinetic cleavage furrow, we propose a previously unrecognized role for AMPK in ensuring that cytokinesis occurs at the proper place and time by establishing a molecular dialog between PLK1 and MRLC in an energy-independent manner.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR ORIENTATION IN THE COLONIAL GREEN ALGA GONIUM PECTORALE (VOLVOCALES)1

Vegetative cells of Gonium pectorale have a fine structure similar to that of Chlamydomonas. In addition, three zones comprise an extracellular matrix; a fibrillar sheath and tripartite boundary surround individual cells, and a fragile capsule zone surrounds the entire colony. Cytokinesis is accomplished by a phycoplast and cleavage furrow. The flagellar apparatus of the immature vegetative cell of this colonial alga is similar to that of Chlamydomonas, but the basal bodies are slightly separated at their proximal ends. The four microtubular rootlets alternate between two and four members. During development, the basal bodies become further separated and nearly parallel. The distal fiber is stretched, but it remains attached to both basal bodies. At maturity, the basal bodies of peripheral cells of the colony have rotated in opposite directions on their longitudinal axes resulting in a displacement of the distal fiber to one side, an asymmetrical orientation of the rootlets and loss of 180° rotational symmetry. Central cells remain similar to Chlamydomonas in that basal bodies do not rotate, rootlets are cruciate, the distal fiber remains medially inserted and 180° rotational symmetry is conserved. A “pin-wheel” configuration of flagellar pairs and the orientation of parallel rootlets toward the colony perimeter probably accounts for the rotation of the colonies during forward swimming. In addition, these ultrastructural features support the traditional placement of G. pectorale as an intermediate between the unicellular Chlamydomonas and the more complex colonial volvocalean genera.     

KLIF-associated cytoskeletal proteins in Trypanosoma brucei regulate cytokinesis by promoting cleavage furrow positioning and ingression

Cytokinesis in the early divergent protozoan Trypanosoma brucei occurs from the anterior cell tip of the new-flagellum daughter toward the nascent posterior end of the old-flagellum daughter of a dividing biflagellated cell. The cleavage furrow ingresses unidirectionally along the preformed cell division fold and is regulated by an orphan kinesin named kinesin localized to the ingressing furrow (KLIF) that localizes to the leading edge of the ingressing furrow. Little is known about how furrow ingression is controlled by KLIF and whether KLIF interacts with and cooperates with other cytokinesis regulatory proteins to promote furrow ingression. Here, we investigated the roles of KLIF in cleavage furrow ingression and identified a cohort of KLIF-associated cytoskeletal proteins as essential cytokinesis regulators. By genetic complementation, we demonstrated the requirement of the kinesin motor activity, but not the putative tropomyosin domain, of KLIF in promoting furrow ingression. We further showed that depletion of KLIF impaired the resolution of the nascent posterior of the old-flagellar daughter cell, thereby stalking cleavage furrow ingression at late stages of cytokinesis. Through proximity biotinylation, we identified a subset of cytoskeleton-associated proteins (CAPs) as KLIF-proximal proteins, and functional characterization of these cytoskeletal proteins revealed the essential roles of CAP46 and CAP52 in positioning the cleavage furrow and the crucial roles of CAP42 and CAP50 in promoting cleavage furrow ingression. Together, these results identified multiple cytoskeletal proteins as cytokinesis regulators and uncovered their essential and distinct roles in cytokinesis.