In this study, we have characterized a novel S. pombe protein, Mid2p, related to the cell division site placement factor, Mid1p. Our results suggest that Mid2p is involved in the establishment of the septin ring that permits efficient cell separation. Moreover, Mid2p degradation may facilitate septin ring disassembly.
Although our data indicate that both Mid1p and Mid2p act during cell division, it appears that their functions are nonoverlapping. There was no synthetic lethality observed in a double deletion strain, only an additive phenotype. Further, Mid1p localized appropriately in the absence of Mid2p and vice versa (unpublished data). Primary amino acid sequence and structure/function analyses also revealed some differences between these proteins. For instance, Mid1p possesses a nuclear localization signal and a large amino-terminal extension not found in Mid2p. On the other hand, there are a number of common elements. The amino-terminal portions of Mid1p and Mid2p contain a domain with significant similarity to the region of Drosophila
anillin shown to directly bind and bundle actin filaments in vitro (Field and Alberts, 1995
), although it remains to be determined whether or not either Mid1p or Mid2p interacts directly with actin filaments. Mid1p and Mid2p both contain PEST motifs conferring protein instability and similar carboxy-terminal regions that include a PH domain (~36%). Although PH domains can mediate the protein–phospholipid interactions necessary to localize proteins to the plasma membrane (for review see Lemmon and Ferguson, 2001
), this region of Mid2p is not sufficient to direct GFP to the cell cortex nor is it required for Mid1p localization to the medial ring (this study; Paoletti and Chang, 2000
). Moreover, the Mid2p PH domain lacks the consensus sequence that predicts direct and avid binding to phosphoinositides (Isakoff et al., 1998
). Thus, the PH domains of these proteins may be involved in protein–protein interactions. In the case of Mid2p, this region is critical for its localization and function, and it is tempting to speculate that it mediates interaction with septins, a hypothesis currently being explored. In the case of Mid1p, the carboxy-terminal region including the PH domain is dispensable for its function (Paoletti and Chang, 2000
). Thus, the amino terminus of Mid1p and the carboxy terminus of Mid2p are likely to be the key determinants in mediating their unique roles during cytokinesis.
As mentioned above, Mid1p and Mid2p share sequence similarity not only with each other but with human anillin. Interestingly, there are multiple uncharacterized anillin homologues in Drosophila
and C. elegans
(Oegema et al., 2000
). The regions of particular sequence similarity include the possible actin-interacting region noted above and an ~200–amino acid stretch that includes the PH domain. Sequence alignments indicate that Mid1p (921 amino acids) is more similar to the larger anillin family members, such as Hs anillin (1,126 residues), Drosophila
anillin (1201 residues), and the C. elegans
gene product Y49E10.19 (1,205 residues). Conversely, Mid2p more closely resembles the smaller gene products, C. elegans
Y43F8C.14 and Drosophila
CG4530. Indeed, the characterized anillin family members in Drosophila
and human seem to be more related to Mid1p than Mid2p in their localization pattern and loss of function phenotypes, with the exception of a connection to septins (Field and Alberts, 1995
; Oegema et al., 2000
). As these anillin homologues in higher eukaryotes are characterized, it will be interesting to learn whether they all function in cytokinesis. Further, it will be interesting to ascertain if they are functionally redundant or have distinct roles during cytokinesis as Mid1p and Mid2p do in S. pombe
Several lines of evidence suggest that Mid2p affects septin ring organization and stability. In the absence of Mid2p, Spn3p–GFP is organized into a loose, rather than a tight, ring that disperses bilaterally across the septum as it forms. Conversely, in cells overproducing a stabilized Mid2p fragment, septin rings, or remnants thereof, persisted one to two cell divisions after they were formed. This observation implies that the turnover of full-length Mid2p might normally permit the timely disassembly of the septin ring. Furthermore, that these cells grew very slowly and were elongated upon the overproduction of nondegradable Mid2p indicates that septin ring disassembly may be important for normal cell cycle progression. In support of this possibility, cells producing a mutant form of the S. cerevisiae
septin, Cdc3p, in which two Cdk sites have been altered to alanine, display two septin rings in G1 (Tang and Reed, 2002
). Significantly, these cells are delayed in cell cycle progression until the old septin ring is disassembled (Tang and Reed, 2002
In an independent study by Berlin et al. (2003)
, further evidence that Mid2p affects septin ring organization was obtained by FRAP analysis. The septin ring was found to be quite stable in wild-type cells, as assessed by the turnover of Spn4p–GFP (F. Chang, personal communication). In contrast, Spn4p–GFP was considerably more dynamic in mid2
Δ cells, indicating that Mid2p function is required for the normal rigidity of the septin ring (F. Chang, personal communication).
Other anillin homologues have also been shown to influence septin ring organization. When C. albicans
Int1p is overproduced in S. cerevisiae
, highly ordered ectopic septin structures are observed in a manner strikingly similar to when Mid2p is overproduced (Gale et al., 2001
). Further, overproduction of a carboxy-terminal fragment of Hs anillin induced the formation of ectopic septin containing structures with which Hs anillin was colocalized (Oegema et al., 2000
). These abnormal cortical foci did not contain either actin or myosin-II (Oegema et al., 2000
), nor did those formed upon Mid2p overproduction ( A; unpublished data). Thus, Mid2p shares with these proteins the ability to interfere with septin organization and function, although it is currently unknown whether the abnormal septin structures produced by Mid2p overproduction contain Mid2p.
An outstanding issue is how the events downstream of septation initiation network activation and septation are restricted temporally. Because all SCF targets identified to date are phosphorylated before being recognized by their cognate F-box protein (Willems et al., 1999
), an appealing hypothesis is that the kinase that presumably modifies Mid2p in order to initiate its destruction might be conditionally activated upon the completion of septation. The identification of the protein kinase that phosphorylates Mid2p would significantly enhance our current understanding of the signaling pathways regulating cell separation.
Because Mid2p is only observed at the site of cell division, it follows then that the destruction machinery must also be recruited there. A candidate F-box protein is Pof6p, which was recently shown to localize to each side of the septum in dividing cells (Hermand et al., 2003
). The loss of function of either Pof6p or the core SCF component Skp1p resulted in aberrant or multiple septa, implying that elevated levels of certain factors may also inhibit efficient cell division (Hermand et al., 2003
). Both the loss of the transcription factor Sep1p (Ribar et al., 1999
) or a slight increase in its abundance generates a cell separation defect (unpublished data). Thus, the proper coordination of cytokinesis appears to require a fine balance between the activation and inhibition of various factors involved in the process.
The loss of function of a wide array of proteins in S. pombe
produces similar cytokinesis defects. For example, deletion of the septins, calcineurin (Ppb1p), a transcription factor (Sep1p), a MAP kinase (Pmk1p), a MAP kinase phosphatase (Pmp1p), members of the exocyst complex (Sec6p), and now Mid2p all cause a long delay in the physical separation of cells after septum synthesis, although the nuclear cycle remains unaffected (J. Pringle, personal communication; Yoshida et al., 1994
; Longtine et al., 1996
; Toda et al., 1996
; Sugiura et al., 1998
; Ribar et al., 1999
; Wang et al., 2002
). Whereas septin ring disorganization is likely to be the underlying cause of the cell separation defect of mid2
Δ cells, this is not the case for ppb1
Δ cells, both Spn3p–GFP and Mid2p–GFP localize normally. This result suggests that even though a number of mutants inhibit cell separation in S. pombe
, defects in multiple pathways may lead to this phenotype. Determining how septin ring organization is affected in other cytokinesis mutants may help elucidate the pathways regulating cell separation.