Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the regulation of fission yeast cytokinesis

Cytokinesis is the last step of the cell-division cycle, which requires precise spatial and temporal regulation to ensure genetic stability. Rho guanine nucleotide exchange factors (Rho GEFs) and Rho GTPases are among the key regulators of cytokinesis. We previously found that putative Rho-GEF Gef2 coordinates with Polo kinase Plo1 to control the medial cortical localization of anillin-like protein Mid1 in fission yeast. Here we show that an adaptor protein, Nod1, colocalizes with Gef2 in the contractile ring and its precursor cortical nodes. Like gef2∆, nod1∆ has strong genetic interactions with various cytokinesis mutants involved in division-site positioning, suggesting a role of Nod1 in early cytokinesis. We find that Nod1 and Gef2 interact through the C-termini, which is important for their localization. The contractile-ring localization of Nod1 and Gef2 also depends on the interaction between Nod1 and the F-BAR protein Cdc15, where the Nod1/Gef2 complex plays a role in contractile-ring maintenance and affects the septation initiation network. Moreover, Gef2 binds to purified GTPases Rho1, Rho4, and Rho5 in vitro. Taken together, our data indicate that Nod1 and Gef2 function cooperatively in a protein complex to regulate fission yeast cytokinesis.     

Molecular organization of cytokinesis node predicts the constriction rate of the contractile ring

The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Characterization of the roles of Blt1p in fission yeast cytokinesis

Spatial and temporal regulation of cytokinesis is essential for cell division, yet the mechanisms that control the formation and constriction of the contractile ring are incompletely understood. In the fission yeast Schizosaccharomyces pombe proteins that contribute to the cytokinetic contractile ring accumulate during interphase in nodes—precursor structures around the equatorial cortex. During mitosis, additional proteins join these nodes, which condense to form the contractile ring. The cytokinesis protein Blt1p is unique in being present continuously in nodes from early interphase through to the contractile ring until cell separation. Blt1p was shown to stabilize interphase nodes, but its functions later in mitosis were unclear. We use analytical ultracentrifugation to show that purified Blt1p is a tetramer. We find that Blt1p interacts physically with Sid2p and Mob1p, a protein kinase complex of the septation initiation network, and confirm known interactions with F-BAR protein Cdc15p. Contractile rings assemble normally in blt1∆ cells, but the initiation of ring constriction and completion of cell division are delayed. We find three defects that likely contribute to this delay. Without Blt1p, contractile rings recruited and retained less Sid2p/Mob1p and Clp1p phosphatase, and β-glucan synthase Bgs1p accumulated slowly at the cleavage site.     

Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis

Cytokinesis is crucial for integrating genome inheritance and cell functions. In multicellular organisms, Rho-guanine nucleotide exchange factors (GEFs) and Rho GTPases are key regulators of division-plane specification and contractile-ring formation during cytokinesis, but how they regulate early steps of cytokinesis in fission yeast remains largely unknown. Here we show that putative Rho-GEF Gef2 and Polo kinase Plo1 coordinate to control the medial cortical localization and function of anillin-related protein Mid1. The division-site positioning defects of gef2Δ plo1-ts18 double mutant can be partially rescued by increasing Mid1 levels. We find that Gef2 physically interacts with the Mid1 N-terminus and modulates Mid1 cortical binding. Gef2 localization to cortical nodes and the contractile ring depends on its last 145 residues, and the DBL-homology domain is important for its function in cytokinesis. Our data suggest the interaction between Rho-GEFs and anillins is an important step in the signaling pathways during cytokinesis. In addition, Gef2 also regulates contractile-ring function late in cytokinesis and may negatively regulate the septation initiation network. Collectively, we propose that Gef2 facilitates and stabilizes Mid1 binding to the medial cortex, where the localized Mid1 specifies the division site and induces contractile-ring assembly.     

Involvement of an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.     

The formins Cdc12 and For3 cooperate during contractile ring assembly in cytokinesis

Both de novo–assembled actin filaments at the division site and existing filaments recruited by directional cortical transport contribute to contractile ring formation during cytokinesis. However, it is unknown which source is more important. Here, we show that fission yeast formin For3 is responsible for node condensation into clumps in the absence of formin Cdc12. For3 localization at the division site depended on the F-BAR protein Cdc15, and for3 deletion was synthetic lethal with mutations that cause defects in contractile ring formation. For3 became essential in cells expressing N-terminal truncations of Cdc12, which were more active in actin assembly but depended on actin filaments for localization to the division site. In tetrad fluorescence microscopy, double mutants of for3 deletion and cdc12 truncations were severely defective in contractile ring assembly and constriction, although cortical transport of actin filaments was normal. Together, these data indicate that different formins cooperate in cytokinesis and that de novo actin assembly at the division site is predominant for contractile ring formation.     

Type II Myosin Heavy Chain Encoded by the myo2 Gene Composes the Contractile Ring during Cytokinesis in Schizosaccharomyces pombe

We cloned the myo2 gene of Schizosaccharomyces pombe, which encodes a type II myosin heavy chain, by virtue of its ability to promote diploidization in fission yeast cells. The myo2 gene encodes 1,526 amino acids in a single open reading frame. Myo2p shows homology to the head domains and the coiledcoil tail of the conventional type II myosin heavy chain and carries putative binding sites for ATP and actin. It also carries the IQ motif, which is a presumed binding site for the myosin light chain. However, Myo2p apparently carries only one IQ motif, while its counterparts in other species have two. There are nine proline residues, which should break α-helix, in the COOH-terminal coiled-coil region of Myo2p. Thus, Myo2p is rather unusual as a type II myosin heavy chain. Disruption of myo2 inhibited cell proliferation. myo2Δ cells showed normal punctate distribution of interphase actin, but they produced irregular actin rings and septa and were impaired in cell separation. Overproduction of Myo2p was also lethal, apparently blocking actin relocation. Nuclear division proceeded without actin ring formation and cytokinesis in cells overexpressing Myo2p, giving rise to multinucleated cells with dumbbell morphology. Analysis using tagged Myo2p revealed that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of the ring and responsible for its contraction. Furthermore, genetic evidence suggested that the acto–myosin system may interact with the Ras pathway, which regulates mating and the maintenance of cell morphology in S. pombe.     

Dephosphorylation of Iqg1 by Cdc14 regulates cytokinesis in budding yeast

Cytokinesis separates cells by contraction of a ring composed of filamentous actin (F-actin) and type II myosin. Iqg1, an IQGAP family member, is an essential protein in Saccharomyces cerevisiae required for assembly and contraction of the actomyosin ring. Localization of F-actin to the ring occurs only after anaphase and is mediated by the calponin homology domain (CHD) of Iqg1, but the regulatory mechanisms that temporally restrict actin ring assembly are not well defined. We tested the hypothesis that dephosphorylation of four perfect cyclin-dependent kinase (Cdk) sites flanking the CHD promotes actin ring formation, using site-specific alanine mutants. Cells expressing the nonphosphorylatable iqg1-4A allele formed actin rings before anaphase and exhibited defects in myosin contraction and cytokinesis. The Cdc14 phosphatase is required for normal cytokinesis and acts on specific Cdk phosphorylation sites. Overexpression of Cdc14 resulted in premature actin ring assembly, whereas inhibition of Cdc14 function prevented actin ring formation. Cdc14 associated with Iqg1, dependent on several CHD-flanking Cdk sites, and efficiently dephosphorylated these sites in vitro. Of importance, the iqg1-4A mutant rescued the inability of cdc14-1 cells to form actin rings. Our data support a model in which dephosphorylation of Cdk sites around the Iqg1 CHD by Cdc14 is both necessary and sufficient to promote actin ring formation. Temporal control of actin ring assembly by Cdk and Cdc14 may help to ensure that cytokinesis onset occurs after nuclear division is complete.     

Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast

In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in Δmid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ∼67% of cells, strands slowly close into rings that constrict without the normal ∼25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.     

Aip1 Promotes Actin Filament Severing by Cofilin and Regulates Constriction of the Cytokinetic Contractile Ring

Aip1 (actin interacting protein 1) is ubiquitous in eukaryotic organisms, where it cooperates with cofilin to disassemble actin filaments, but neither its mechanism of action nor its biological functions have been clear. We purified both fission yeast and human Aip1 and investigated their biochemical activities with or without cofilin. Both types of Aip1 bind actin filaments with micromolar affinities and weakly nucleate actin polymerization. Aip1 increases up to 12-fold the rate that high concentrations of yeast or human cofilin sever actin filaments, most likely by competing with cofilin for binding to the side of actin filaments, reducing the occupancy of the filaments by cofilin to a range favorable for severing. Aip1 does not cap the barbed ends of filaments severed by cofilin. Fission yeast lacking Aip1 are viable and assemble cytokinetic contractile rings normally, but rings in these Δaip1 cells accumulate 30% less myosin II. Further, these mutant cells initiate the ingression of cleavage furrows earlier than normal, shortening the stage of cytokinetic ring maturation by 50%. The Δaip1 mutation has negative genetic interactions with deletion mutations of both capping protein subunits and cofilin mutations with severing defects, but no genetic interaction with deletion of coronin.     

The novel proteins Rng8 and Rng9 regulate the myosin-V Myo51 during fission yeast cytokinesis

The myosin-V family of molecular motors is known to be under sophisticated regulation, but our knowledge of the roles and regulation of myosin-Vs in cytokinesis is limited. Here, we report that the myosin-V Myo51 affects contractile ring assembly and stability during fission yeast cytokinesis, and is regulated by two novel coiled-coil proteins, Rng8 and Rng9. Both rng8Δ and rng9Δ cells display similar defects as myo51Δ in cytokinesis. Rng8 and Rng9 are required for Myo51’s localizations to cytoplasmic puncta, actin cables, and the contractile ring. Myo51 puncta contain multiple Myo51 molecules and walk continuously on actin filaments in rng8⁺ cells, whereas Myo51 forms speckles containing only one dimer and does not move efficiently on actin tracks in rng8Δ. Consistently, Myo51 transports artificial cargos efficiently in vivo, and this activity is regulated by Rng8. Purified Rng8 and Rng9 form stable higher-order complexes. Collectively, we propose that Rng8 and Rng9 form oligomers and cluster multiple Myo51 dimers to regulate Myo51 localization and functions.     

Gef1p and Scd1p,the Two GDP-GTP exchange factors for Cdc42p,form a ring structure that shrinks during cytokinesis in Schizosaccharomyces pombe

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.     

A Highlights from MBoC Selection: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

Myosin?II heavy chain and formin mediate the targeting of myosin essential light chain to the division site before and during cytokinesis

MLC1 is a haploinsufficient gene encoding the essential light chain for Myo1, the sole myosin‑II heavy chain in the budding yeast Saccharomyces cerevisiae. Mlc1 defines an essential hub that coordinates actomyosin ring function, membrane trafficking, and septum formation during cytokinesis by binding to IQGAP, myosin‑II, and myosin‑V. However, the mechanism of how Mlc1 is targeted to the division site during the cell cycle remains unsolved. By constructing a GFP‑tagged MLC1 under its own promoter control and using quantitative live‑cell imaging coupled with yeast mutants, we found that septin ring and actin filaments mediate the targeting of Mlc1 to the division site before and during cytokinesis, respectively. Both mechanisms contribute to and are collectively required for the accumulation of Mlc1 at the division site during cytokinesis. We also found that Myo1 plays a major role in the septin‑dependent Mlc1 localization before cytokinesis, whereas the formin Bni1 plays a major role in the actin filament–dependent Mlc1 localization during cytokinesis. Such a two‑tiered mechanism for Mlc1 localization is presumably required for the ordered assembly and robustness of cytokinesis machinery and is likely conserved across species.     

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15_ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.     

Actin filament severing by cofilin is more important for assembly than constriction of the cytokinetic contractile ring

We created two new mutants of fission yeast cofilin to investigate why cytokinesis in many organisms depends on this small actin-binding protein. These mutant cofilins bound actin monomers normally, but bound and severed ADP-actin filaments much slower than wild-type cofilin. Cells depending on mutant cofilins condensed nodes, precursors of the contractile ring, into clumps rather than rings. Starting from clumped nodes, mutant cells slowly assembled rings from diverse intermediate structures including spiral strands containing actin filaments and other contractile ring proteins. This process in mutant cells depended on α-actinin. These slowly assembled contractile rings constricted at a normal rate but with more variability, indicating ring constriction is not very sensitive to defects in severing by cofilin. Computer simulations of the search-capture-pull and release model of contractile ring formation predicted that nodes clump when the release step is slow, so cofilin severing of actin filament connections between nodes likely contributes to the release step.     

Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.     

Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase

We investigated the assembly of cortical nodes that generate the cytokinetic contractile ring in fission yeast. Observations of cells expressing fluorescent fusion proteins revealed two types of interphase nodes. Type 1 nodes containing kinase Cdr1p, kinase Cdr2p, and anillin Mid1p form in the cortex around the nucleus early in G2. Type 2 nodes with protein Blt1p, guanosine triphosphate exchange factor Gef2p, and kinesin Klp8p emerge from contractile ring remnants. Quantitative measurements and computer simulations showed that these two types of nodes come together by a diffuse-and-capture mechanism: type 2 nodes diffuse to the equator and are captured by stationary type 1 nodes. During mitosis, cytokinetic nodes with Mid1p and all of the type 2 node markers incorporate into the contractile ring, whereas type 1 nodes with Cdr1p and Cdr2p follow the separating nuclei before dispersing into the cytoplasm, dependent on septation initiation network signaling. The two types of interphase nodes follow parallel branches of the pathway to prepare nodes for cytokinesis.