Myosin-II is mobile at the division site during the early part of the cell cycle and becomes progressively immobilized from anaphase to the onset of cytokinesis
To determine the dynamics of the AMR components, we first analyzed the dynamics of Myo1, the sole myosin-II heavy chain in budding yeast, during the cell cycle. When the entire Myo1-GFP ring at the bud neck was photobleached, the mean of the maximal fluorescence recovery was <8% regardless of cell cycle stages (Fig. S1, A–C
; Video 1
; and the entire FRAP data with individual curves and quantitative analyses were also shown in Figs. S3
), which are marked by bud size (see Materials and methods for details) and septin–hourglass splitting (see associated videos; not depicted in figures), a cellular event that coincides with the onset of cytokinesis (Lippincott et al., 2001
). The recovery was noticeably higher in small-budded cells (7.2 ± 1.1%) than in cells undergoing cytokinesis (1.1 ± 1.3%). These FRAP data suggest that there is a limited exchange of Myo1 between the bud neck and the cytosol throughout the cell cycle, which could be caused by a slow rate of exchange and/or a small pool of Myo1 in the cytosol.
As a complementary approach to FRAP, we also used fluorescence loss in photobleaching (FLIP) to probe the dynamics of Myo1 at the bud neck. A cytosolic region of a mother cell with a small bud was photobleached sequentially four times (Fig. S1 D). The fluorescence intensity of Myo1-GFP at the bud neck decreased only 30% in 23 min after the initial bleaching in the cytosol, in comparison to a 20% fluorescence loss in an unbleached control cell in the same imaging field during the same period (Fig. S1 E). In contrast, when a similar experiment was performed on a cell carrying Tpm2-GFP (tropomyosin), which is highly dynamic (see ), the fluorescence intensity of Tpm2 at the bud neck decreased 50% in 50 s even after one bleach in the cytosol, whereas little or no change was observed for the control cell in the same field during the same period (Fig. S1, F–H). These FLIP data suggest that Myo1 cycles between the bud neck and the cytosol slowly. We also monitored the kinetics of Myo1 localization during the cell cycle, and found that Myo1 signal at the bud neck reached its peak from bud emergence to the small-budded stage (in <30 min; unpublished data). From this point on, Myo1 remained fairly constant at the bud neck (Fig. S1, I and J). Approximately 20 min before septin–hourglass splitting, Myo1 intensity was briefly increased by ~20% and then decreased in a linear fashion during AMR constriction (Fig. S1, I and J; Tully et al., 2009
). Direct measurement of Myo1-GFP (the sole source of Myo1 expressed from its native promoter at its physiological locus) showed that nearly all of the Myo1 molecules were localized to the bud neck around the small-budded stage. Because of the very dim signal of Myo1-GFP in the cytosol, it was rather difficult to obtain an accurate and meaningful measurement of Myo1 in this pool. However, based on fluorescence recovery after full-ring bleaching (Fig. S1 A) and the measurement of Myo1 intensity at the bud neck throughout the cell cycle (Fig. S1, I and J), we estimate that ≥80% of the total cellular Myo1 is localized to the bud neck before cytokinesis. Together, these data indicate that the majority of Myo1 are localized to the division site early in the cell cycle and maintained there with little flux between the ring and the cytosol.
Figure 5. Actin ring–associated proteins and membrane trafficking components are dynamic during cytokinesis. (A) Tropomyosin is highly dynamic during cytokinesis. The full ring of Tpm2-GFP from a cell of the strain YEF6197 (TPM2-GFP CDC3-RFP) undergoing (more ...)
To determine Myo1 dynamics within the ring structure at the bud neck, we bleached a half of the ring in cells at different stages of the cell cycle. In small-budded cells, Myo1-GFP signal in the bleached region recovered quickly with a recovery rate (t1/2
) of 20.4 ± 1.3 s and a maximal recovery of 19.3 ± 0.8% (, solid circles; and Video 2
, left), whereas the GFP signal in the unbleached region decreased correspondingly (, open circles), suggesting that Myo1 moves laterally from the unbleached region to the bleached region. The magnitude of recovery is significant, considering that the maximally possible recovery would be 50% if all the recovery in the bleached half were attributed to lateral movement within the ring structure. This is a reasonable assumption given that Myo1 displays little flux between the ring and the cytosol (see the preceding paragraphs). In large-budded cells, the maximal recovery was limited to 7.1 ± 1.1% ( and Video 2, middle). In cells undergoing cytokinesis, virtually no recovery was observed ( and Video 2, right). Thus, Myo1 is mobile within the ring structure during the early part of the cell cycle and becomes immobilized toward the late part of the cell cycle.
Figure 1. Myo1 displays cell cycle–regulated dynamics at the division site. (A–C) Myo1 is mobile at the division site in small-budded cells (A), becomes less mobile in large-budded cells (B), and is immobilized during cytokinesis (C), indicated (more ...)
To determine the transition point in Myo1 mobility within the ring structure more precisely, we performed similar FRAP analysis on cells carrying mCherry-labeled Nup57, a component of the nuclear pore complex (Alber et al., 2007
) that marks the nuclear position in the cell. In wild-type cells, the penetration of the nucleus from the mother into the daughter cell compartment is correlated with the onset of anaphase (Yang et al., 1997
). We found that Myo1 became increasingly immobile from middle or late anaphase to the onset of cytokinesis (). In addition, Myo1 remained highly mobile in mutants arrested at the onset of anaphase (cdc16
) and was nearly immobile in mutants arrested at the mitotic exit (cdc15-2
and dbf2-1 dbf20Δ
; unpublished data). Together, these data indicate that Myo1 is mobile within the ring structure before the onset of anaphase, increasingly immobilized from anaphase to telophase, and becomes completely immobile during cytokinesis.
We then probed the dynamics of the regulatory light chain (RLC) and the essential light chain (ELC) for Myo1. Mlc2, the RLC for Myo1, displays a localization pattern identical to Myo1 throughout the cell cycle, and its localization to the bud neck completely depends on its binding to Myo1 (Luo et al., 2004
). Mlc2-GFP showed nearly identical dynamics as Myo1-GFP during the cell cycle when either the entire ring (not depicted) or a half of the ring (Fig. S2, A–C
) was bleached.
Mlc1, the ELC for Myo1 (Luo et al., 2004
), also binds to Myo2 (a myosin-V in budding yeast) and Iqg1 (the sole IQGAP in budding yeast) via their respective IQ motifs (Stevens and Davis, 1998
; Boyne et al., 2000
; Shannon and Li, 2000
). In small-budded cells, Mlc1 localizes to the bud cortex as puncta, reflecting its association with myosin-V and secretory vesicles (Wagner et al., 2002
; Luo et al., 2004
). These puncta were highly dynamic (Fig. S2 D). Mlc1 localizes to the bud neck only in large-budded cells and cells undergoing cytokinesis (Shannon and Li, 2000
; Wagner et al., 2002
; Luo et al., 2004
). In these cells, Mlc1 displayed immobility from its initial localization to the completion of cytokinesis (see and Video 8
). Together, these data indicate that myosin-II undergoes cell cycle–regulated changes in dynamics, being mobile within the ring structure early in the cell cycle and becoming progressively immobilized from anaphase to the onset of cytokinesis.
Figure 7. Proteins involved in septum formation or its coordination with the AMR display Myo1-dependent immobility during cytokinesis. (A–D) The immobility of Mlc1, Iqg1, Inn1, and Hof1 during cytokinesis depends on Myo1. FRAP analysis was performed on (more ...)
The change in Myo1 dynamics is also reflected by the dynamics of its binding partners during the cell cycle. Bni5, a septin-binding protein (Lee et al., 2002
), mediates Myo1 targeting to the division site from late G1 to the onset of telophase, whereas Mlc1 and Iqg1 mediate Myo1 targeting from the onset of anaphase to the end of cytokinesis (Fang et al., 2010
). Not surprisingly, Bni5 was mobile at the division site during its entire stay in the presence or absence of Myo1 (Fig. S2, E–H; and Video 3
, left); in contrast, Mlc1 (see and Video 8) and Iqg1 (see ) displayed immobility throughout their localization at the division site.
Actin filaments, motor domain, and light chain binding sites are not required for Myo1 immobility during cytokinesis
The timing of Myo1 mobility changes during the cell cycle correlates with the timing of actin ring assembly and AMR constriction (Epp and Chant, 1997
; Bi et al., 1998
; Lippincott and Li, 1998a
). To determine the possible role of actin ring assembly in regulating Myo1 immobility, we examined Myo1 dynamics in bni1Δ
cells during the cell cycle. Bni1 and Bnr1 are two formins in budding yeast that share an essential role in nucleating actin cable assembly to mediate polarized cell growth during budding (Pruyne et al., 2002
; Sagot et al., 2002
). During cytokinesis, Bni1 is the only formin localized at the division site (Buttery et al., 2007
) and plays an important role in actin ring assembly and cytokinesis (Vallen et al., 2000
; Tolliday et al., 2002
). We found that deletion of BNI1
did not affect Myo1 dynamics during the cell cycle, including its immobility during cytokinesis (a cell in which Myo1-GFP failed to constrict presumably because of the absence of the actin ring; and Video 3, right). Because about one third of bni1Δ
cells are still able to form a faint actin ring, we disrupted all actin filaments using latrunculin A (LatA; Ayscough et al., 1997
) and then examined Myo1 dynamics. The LatA treatment did not affect Myo1 behavior during the cell cycle (; and Video 3, right). Myo1 was mobile at the division site in small-budded cells (t1/2
, 27.8 ± 7.5; maximal recovery, 18.6 ± 3.7%; n
= 6), less mobile in large-budded cells (maximal recovery, 6.9 ± 1.2%; n
= 5), and became immobile during cytokinesis (n
= 6). As expected, Myo1-GFP failed to constrict in LatA-treated cells ( and Video 3, right), in contrast to the DMSO-treated control cell (). These data indicate that the actin ring and thus AMR constriction are not required for Myo1 immobility during cytokinesis.
Figure 2. Actin filaments are not required for Myo1 immobility during cytokinesis. (A) Myo1 remains immobile during cytokinesis in bni1Δ cells. Myo1-GFP ring in a cell of the strain YEF6116 (bni1Δ MYO1-GFP CDC3-RFP) undergoing cytokinesis was analyzed (more ...)
The tail of Myo1 is sufficient for directing the assembly of a “headless” AMR (Fang et al., 2010
), which largely fulfills its role in cytokinesis (Lord et al., 2005
; Fang et al., 2010
). However, this headless AMR constricts with ~70–80% of the constriction rate of a normal AMR (Lord et al., 2005
; Fang et al., 2010
). To determine whether the head domain of Myo1, which includes its motor domain, a putative actin binding site, and the binding sites for both ELC and RLC, plays any role in regulating its immobility during cytokinesis, we performed FRAP analysis on yeast cells in which the chromosomal copy of MYO1
was precisely replaced with the Myo1 tail (residues 856–1,928)–coding sequence (Fang et al., 2010
). Surprisingly, the dynamic behavior of the Myo1 tail was very similar to that of the full-length protein, being mobile at the division site in small-budded cells (t1/2
, 22.7 ± 3.2; maximal recovery, 27.3 ± 2.8%; n
= 12; and Video 4
, left) and less mobile in large-budded cells (maximal recovery, 12.4 ± 1.6%; n
= 7; and Video 4, middle) but becoming immobilized during cytokinesis (n
= 7; and Video 4, right). Thus, the regulation of Myo1 dynamics during the cell cycle is largely mediated by its tail not its head domain.
Figure 3. The tail of Myo1 confers its dynamic property during the cell cycle. (A–C) Myo1-Tail-GFP in a small-budded cell (A), a large-budded cell (B), and a cell undergoing cytokinesis (C) of the strain XDY288 (myo1-Tail-GFP CDC3-RFP) was analyzed by FRAP. (more ...)
A small region near the C terminus of Myo1 is required for its immobility during cytokinesis
All myosin-IIs from animal cells can assemble into bipolar filaments in vitro, and this assembly invariably depends on a region near their C termini, called the assembly domain, which is required for antiparallel interaction of myosin-II molecules (Trybus, 1991
; Tan et al., 1992
). Myo1 tail also contains a putative assembly domain near its C terminus, which can only localize to the division site if it coexists with a Myo1 molecule harboring the targeting domains (mTD1 and TD2) near the middle of its tail (; Fang et al., 2010
). Strikingly, seven of the 10 point mutations in MYO1
that are synthetically lethal with the deletion of HOF1
(Nishihama et al., 2009
), which encodes an F-BAR protein involved in cytokinesis (Kamei et al., 1998
; Lippincott and Li, 1998b
; Vallen et al., 2000
), are clustered within or near the putative assembly domain, highlighting the importance of this region in Myo1 function and cytokinesis (). Six of the seven are stop codon mutations, which define five distinct truncation alleles of MYO1
(). Based on these observations, we hypothesized that Myo1 may undergo cell cycle–triggered higher-order assembly, forming bipolar filaments during cytokinesis that account for its immobility.
Figure 4. A small C-terminal region of Myo1 is required for its immobility during cytokinesis. (A) Myo1 motifs and the positions of myo1 mutations that are synthetically with hof1Δ. (B and C) Myo1 dynamics are not affected by the deletion of its putative (more ...)
To test this hypothesis, we performed FRAP analysis on cells carrying one of four truncation alleles of MYO1
(stopped at residue 1,483, 1,535, 1,633, or 1,798) isolated from the hof1
synthetic lethal screen as well as a truncation allele deleted for the coding sequence of a smaller C-terminal region (stopped at residue 1,903), including the predicted nonhelical tailpiece (). Similar nonhelical regions have been implicated in filament assembly for smooth muscle and nonmuscle myosin-IIs (Trybus, 1991
). Like the wild-type protein, Myo1 lacking the putative nonhelical region was immobile during cytokinesis, as indicated by the half-ring bleaching ( and Video 5
, left), and virtually no recovery was observed when the entire ring was bleached (). Thus, the putative nonhelical region of Myo1 is not required for its immobility during cytokinesis. In contrast, Myo1-(AA1–1798) was mobile at the division site during cytokinesis (t1/2
, 12.7 ± 3.4; maximal recovery, 18.3 ± 2.1%; n
= 8; and Video 5, right), even though its expression level was similar to that of the full-length protein or Myo1 lacking the putative nonhelical region (). In addition, when the entire ring was bleached during cytokinesis, the recovery was noticeably higher than that of the full-length protein ( compare with Fig. S1 C). The other three Myo1 variants with larger truncations also displayed mobility, although their overall signal at the bud neck was dimmer than the full-length or Myo1-(AA1–1798) proteins during cytokinesis (unpublished data). Together, these data demonstrate that a 105-aa fragment (residues 1,798–1,903) near the C terminus of Myo1 is essential for the establishment and/or maintenance of its immobility during cytokinesis.
Actin ring–associated proteins and membrane trafficking components are dynamic during cytokinesis
To gain insight into the construction of the cytokinesis machinery, we compared the dynamics of Myo1 and other cytokinesis proteins by performing FRAP analysis on those proteins involved in actin ring assembly, membrane trafficking, and septum formation. Because GFP-tagged actin is not functional and does not label actin cables or the actin ring (Doyle and Botstein, 1996
), we probed the dynamics of actin ring–associated proteins (formins and tropomyosin) instead of actin itself. Both formin and tropomyosin are universally required for nucleating and stabilizing the actin filaments in the AMR (Balasubramanian et al., 2004
; Moseley and Goode, 2006
; Pollard, 2008
). The formin Bni1 localizes to the sites of polarized growth during the cell cycle, including the bud neck during cytokinesis, whereas the formin Bnr1 localizes to the mother side of the bud neck from bud emergence to the onset of cytokinesis (Pruyne et al., 2004
; Buttery et al., 2007
). Thus, Bni1 is the only formin associated with the actin ring during cytokinesis. FRAP analysis indicates that Bni1-3GFP, expressed from its native promoter, is dynamic at the bud cortex as well as at the bud neck (Buttery et al., 2007
). In contrast, the neck-localized Bnr1-GFP, expressed from its native promoter, is relatively immobile (Buttery et al., 2007
). These observations are essentially confirmed in our study. In our experiments, GFP-tagged Bni1, expressed from its native promoter and carried on a high-copy plasmid (a single GFP-tagged Bni1 did not produce a signal strong enough for our study), localized to the division site at the onset of cytokinesis and was highly dynamic (Fig. S2 J). After full-ring bleaching, Bni1 displayed a recovery rate (t1/2
) of 14.6 ± 1.3 s and a maximal recovery of 31.1 ± 1.9%. As expected, GFP-tagged Bnr1, expressed from a methionine promoter carried on a plasmid, was largely immobile throughout the cell cycle (Fig. S2 I).
GFP-tagged tropomyosins have been used to label the actin ring in live cells of the budding and fission yeasts (Pelham and Chang, 2002
; Yoshida et al., 2006
). We found that GFP-tagged Tpm2 localized to the division site 1–2 min before the onset of cytokinesis and was highly dynamic, with a recovery rate (t1/2
) of 2.0 ± 0.5 s and a maximal recovery of 18.6 ± 2.9% ( and Video 6
, left). Together, these data indicate that actin ring–associated proteins, in contrast to myosin-II, are dynamic at the bud neck throughout cytokinesis.
To determine the dynamics of membrane trafficking components during cytokinesis, we performed FRAP analysis on cells carrying GFP-tagged Myo2 (myosin-V) and Exo84 (a subunit of the exocyst). Myo2 is required for the transport of post-Golgi vesicles along actin cables to the sites of polarized growth, including the bud neck during cytokinesis (Bretscher, 2003
). Exo84 is a vesicle-associated subunit of the exocyst that is required for the tethering of post-Golgi vesicles to the PM during polarized cell growth and cytokinesis (Guo et al., 2000
; Boyd et al., 2004
). Both Myo2 and Exo84 arrive at the division site around the onset of cytokinesis (Fang et al., 2010
; Wloka et al., 2011
). Upon full-ring bleaching, Myo2-GFP recovered with a fast rate (t1/2
) of 11.0 ± 1.3 s and a maximal recovery of 47.1 ± 5.0% ( and Video 6, middle) throughout cytokinesis (AMR constriction was marked by RFP-tagged Myo1 in the same cells). Similarly, Exo84 recovered with a fast rate (t1/2
) of 12.2 ± 0.6 s and a maximal recovery of 35.9 ± 2.0% ( and Video 6, right; Boyd et al., 2004
). The dynamic properties of Myo2 and Exo84 are consistent with their role in membrane trafficking during cytokinesis.
Proteins involved in septum formation or its coordination with the AMR display Myo1-dependent immobility during cytokinesis
Chs2, the chitin synthase II, is delivered to the bud neck at the onset of cytokinesis by the exocytic machinery to execute its essential role in PS formation (Sburlati and Cabib, 1986
; Chuang and Schekman, 1996
; VerPlank and Li, 2005
). Bleaching of the full (unpublished data)- or half-ring of Chs2-GFP within the first 3–4 min after its localization to the bud neck led to a full recovery ( and Video 7
), which presumably reflects its timed and continuous delivery to the division site by the exocytic machinery. After this period, Chs2 became immobile ( and Video 7). Together, these results suggest that (a) Chs2 delivery to the division site is complete within a few minutes of its initial localization; (b) once delivered, Chs2 may be closely associated with Myo1; and (c) Chs2 is not recycled back to the furrow membrane after its endocytic removal during the late stage of cytokinesis. Thus, Chs2 displays biphasic dynamics during cytokinesis.
Figure 6. Chs2 displays biphasic dynamics during cytokinesis, and its immobility depends on Myo1. (A) Chs2 is dynamic and then immobile during cytokinesis. Chs2-GFP in the strain YEF5874 (CHS2-GFP CDC3-RFP) was bleached sequentially during cytokinesis. Time 0 corresponds (more ...)
To determine whether the immobility of Chs2 during cytokinesis depends on Myo1, we examined Chs2 dynamics in myo1Δ cells. Strikingly, Chs2 became mobile throughout cytokinesis and, as expected, failed to constrict ( and Video 7), suggesting that Chs2 is immobilized in a Myo1-dependent manner.
To determine whether Myo1 and other major components of the division machinery act in unison during cytokinesis such that deletion of one component will change the organization and/or the dynamics of other components, we examined Myo1 dynamics during cytokinesis in several cytokinesis mutants. In chs2Δ
cells, in which PS formation is completely blocked and cytokinesis is more defective than in myo1Δ
cells (Bi, 2001
; Schmidt et al., 2002
; VerPlank and Li, 2005
; Sanchez-Diaz et al., 2008
; Nishihama et al., 2009
; Meitinger et al., 2010
), Myo1 remained immobile (; and Video 7). The Myo1 immobility was also observed in mlc2Δ
, and cyk3Δ
cells (unpublished data). Myo1 dynamics during cytokinesis could not be probed in mlc1Δ
cells, as both Mlc1 and Iqg1 are required for Myo1 targeting to the division site during cytokinesis (Fang et al., 2010
), and mlc1Δ
are lethal in most strain backgrounds under normal growth conditions (Epp and Chant, 1997
; Stevens and Davis, 1998
). Together, these data indicate that the immobility of Chs2 during cytokinesis, but not its delivery to the division site, depends on Myo1; in contrast, Myo1 immobility does not depend on any other aforementioned cytokinesis proteins.
Besides Chs2, several other proteins (Mlc1, Iqg1, Inn1, Hof1, and Cyk3) have been implicated in PS formation or its coordination with the AMR (Korinek et al., 2000
; Wagner et al., 2002
; Nishihama et al., 2009
; Meitinger et al., 2010
). To determine their dynamics and explore their relationships with Myo1 during cytokinesis, we performed FRAP analysis of these proteins in wild-type and myo1Δ
cells. As described earlier, in a wild-type strain, Mlc1, the ELC for Myo1, was immobile upon its localization to the bud neck in large-budded cells and remained immobile during cytokinesis (, left; and Video 8). However, in myo1Δ
cells, Mlc1 became dynamic (, right; and Video 8). Both half-ring and full-ring (, arrowhead) bleaching indicates that the recovery is largely caused by cytosol–neck exchange. Like Mlc1, Iqg1 was immobile during cytokinesis (, left; and Video 8) but became dynamic in myo1Δ
cells (, right; and Video 8). These data suggest that even though Mlc1 and Iqg1 are recruited to the neck in a Myo1-independent fashion and are actually required for Myo1 localization at the division site during cytokinesis (Fang et al., 2010
). Myo1, in turn, is required for their organization.
Inn1 plays an essential role in PS formation and interacts with the SH3 domain of the F-BAR protein Hof1 and of the transglutaminase domain–containing protein Cyk3 through distinct PXXP motifs in its C terminus (Nishihama et al., 2009
). Both Hof1 and Cyk3 are also involved in PS formation (Korinek et al., 2000
; Vallen et al., 2000
; Meitinger et al., 2010
). Inn1 localized to the bud neck at the onset of cytokinesis (Sanchez-Diaz et al., 2008
; Nishihama et al., 2009
) and remained immobile throughout the division process (, left; and Video 9
). However, in myo1Δ
cells, Inn1 became more dynamic (, right; and Video 9). Hof1 was relatively dynamic until the onset of cytokinesis when it quickly became immobile and remained so during cytokinesis (, left; Video 9; and not depicted). Hof1 became much more dynamic in myo1Δ
cells (, right; and Video 9). Thus, the immobility of Inn1 and Hof1 depends on Myo1. Interestingly, Cyk3 was dynamic throughout cytokinesis (t1/2
of 7.4 ± 0.9 s and maximal recovery of 31.7 ± 1.9% after full-ring bleaching; Fig. S2 K). These results suggest that Inn1 and Hof1 are closely associated with Myo1 during cytokinesis, whereas Cyk3 is fluxing between the bud neck and the cytosol. Together, our data indicate that proteins involved in PS formation, with the exception of Cyk3, display Myo1-dependent immobility during cytokinesis.
Because Myo1 immobility depends on a small region near its C-terminal end, we examined Chs2 and Hof1 dynamics in cells carrying the myo1-(AA1798Stop)
allele (compare with ). Both Chs2 ( and Video 10
, left) and Hof1 ( and Video 10, right) became mobile at the division site during cytokinesis in the majority of the cells examined. We also probed the dynamics of Chs2 and Hof1 in cells carrying the myo1-(AA1535Stop)
allele (compare with ). In this case, Chs2 and Hof1 were mobile in all cells examined (unpublished data). Thus, the immobility of Chs2 and Hof1 depends on the immobility, not just the presence, of Myo1 at the division site.
Figure 8. The immobility of Chs2 and Hof1 depends on a small region near the C-terminal end of Myo1. (A and B) Chs2 and Hof1 become mobile at the division site during cytokinesis in myo1-(AA1798Stop) cells. Chs2-GFP in strain YEF6771 (myo1-(AA1798Stop) CHS2-GFP (more ...)