Role of Inn1 and its interactions with Hof1 and Cyk3 in promoting cleavage furrow and septum formation in S. cerevisiae

Cytokinesis requires coordination of actomyosin ring (AMR) contraction with rearrangements of the plasma membrane and extracellular matrix. In Saccharomyces cerevisiae, new membrane, the chitin synthase Chs2 (which forms the primary septum [PS]), and the protein Inn1 are all delivered to the division site upon mitotic exit even when the AMR is absent. Inn1 is essential for PS formation but not for Chs2 localization. The Inn1 C-terminal region is necessary for localization, and distinct PXXP motifs in this region mediate functionally important interactions with SH3 domains in the cytokinesis proteins Hof1 (an F-BAR protein) and Cyk3 (whose overexpression can restore PS formation in inn1Δ cells). The Inn1 N terminus resembles C2 domains but does not appear to bind phospholipids; nonetheless, when overexpressed or fused to Hof1, it can provide Inn1 function even in the absence of the AMR. Thus, Inn1 and Cyk3 appear to cooperate in activating Chs2 for PS formation, which allows coordination of AMR contraction with ingression of the cleavage furrow

Investigation of phospholipid effector domains

Phospholipid-binding modules such as PH, C1 and C2 domains play crucial roles in location-dependent regulation of their host proteins and are important many cellular processes. The work described in this dissertation comprises the screen for undescribed phospholipid effector domains and a more detailed biochemical analysis of two classes of domains that were identified in this screen. We identify the KA1 domain (kinase associated domain-1), found at the C-terminus of yeast septin-associated kinases (Kcc4p, Gin4p and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Using X-ray crystallography, we show that there is a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins, and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to \u27coincidence detection\u27, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer\u27s disease, cancer, and autism. We also investigate the in vitro and in vivo membrane binding properties of three S. cerevisiae F-BAR domains, which are known to sense/stabilize membrane curvature in cellular processes where membranes are going through major shape changes. We show that the F-BAR domain from Rho GTPase activating protein Rgd1p preferentially associates with phosphoinositides, whereas the F-BAR domains from adaptor proteins Bzz1p and Hof1p bind similarly to different phospholipids. We describe the X-ray crystal structures of Hof1p and ligand-bound Rgd1p F-BAR domains and propose a model of phosphoinositide recognition based on a specific positively charged site found in a subset of F-BAR proteins. The studies described here have significant implications for understanding the F-BAR domain proteins and the vital cellular processes they are involved in. From biomedical perspective, understanding of F-BAR domains is critical since F-BAR domain proteins are implicated in Huntington\u27s disease, cancer and auto- inflammatory disease

Investigation of phospholipid effector domains

Phospholipid-binding modules such as PH, C1 and C2 domains play crucial roles in location-dependent regulation of their host proteins and are important many cellular processes. The work described in this dissertation comprises the screen for undescribed phospholipid effector domains and a more detailed biochemical analysis of two classes of domains that were identified in this screen. We identify the KA1 domain (kinase associated domain-1), found at the C-terminus of yeast septin-associated kinases (Kcc4p, Gin4p and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Using X-ray crystallography, we show that there is a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins, and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to \u27coincidence detection\u27, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer\u27s disease, cancer, and autism. We also investigate the in vitro and in vivo membrane binding properties of three S. cerevisiae F-BAR domains, which are known to sense/stabilize membrane curvature in cellular processes where membranes are going through major shape changes. We show that the F-BAR domain from Rho GTPase activating protein Rgd1p preferentially associates with phosphoinositides, whereas the F-BAR domains from adaptor proteins Bzz1p and Hof1p bind similarly to different phospholipids. We describe the X-ray crystal structures of Hof1p and ligand-bound Rgd1p F-BAR domains and propose a model of phosphoinositide recognition based on a specific positively charged site found in a subset of F-BAR proteins. The studies described here have significant implications for understanding the F-BAR domain proteins and the vital cellular processes they are involved in. From biomedical perspective, understanding of F-BAR domains is critical since F-BAR domain proteins are implicated in Huntington\u27s disease, cancer and auto- inflammatory disease

<i>Drosophila</i> TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

<div><p>Regulated nuclear entry of clock proteins is a conserved feature of eukaryotic circadian clocks and serves to separate the phase of mRNA activation from mRNA repression in the molecular feedback loop. In <i>Drosophila</i>, nuclear entry of the clock proteins, PERIOD (PER) and TIMELESS (TIM), is tightly controlled, and impairments of this process produce profound behavioral phenotypes. We report here that nuclear entry of PER-TIM in clock cells, and consequently behavioral rhythms, require a specific member of a classic nuclear import pathway, Importin α1 (IMPα1). In addition to IMPα1, rhythmic behavior and nuclear expression of PER-TIM require a specific nuclear pore protein, Nup153, and Ran-GTPase. IMPα1 can also drive rapid and efficient nuclear expression of TIM and PER in cultured cells, although the effect on PER is mediated by TIM. Mapping of interaction domains between IMPα1 and TIM/PER suggests that TIM is the primary cargo for the importin machinery. This is supported by attenuated interaction of IMPα1 with TIM carrying a mutation previously shown to prevent nuclear entry of TIM and PER. TIM is detected at the nuclear envelope, and computational modeling suggests that it contains HEAT-ARM repeats typically found in karyopherins, consistent with its role as a co-transporter for PER. These findings suggest that although PER is the major timekeeper of the clock, TIM is the primary target of nuclear import mechanisms. Thus, the circadian clock uses specific components of the importin pathway with a novel twist in that TIM serves a karyopherin-like role for PER.</p></div

Downregulation of IMPα1 in clock neurons disrupts rest:activity rhythms and nuclear translocation of clock proteins, PER and TIM.

<p>(A) IMPα1 knockdown leads to arrhythmia in constant darkness (DD). Genotypes are indicated on the top of each panel. The gray and black bars indicate subjective day and night, respectively. Representative activity records are shown. (B) Downregulation of IMPα1 impairs nuclear translocation of clock proteins, PER and TIM. Control flies and IMPα1 knockdown flies were collected at ZT0 and brains were dissected and stained with antibodies to PER or TIM (green) and PDF (red).</p

TIM is primary cargo for IMPα1.

<p>(A) S2 cells were transiently transfected with pIZ-<i>tim</i>-V5 (wt), pIZ-<i>tim</i>_mNLS-V5 (<i>tim</i> carrying a mutant NLS), pIZ-<i>imp_α1</i>-VSV, or pIZ-<i>imp_α1</i>∆IBB-VSV as indicated. After 60 hours, cell lysates were immunoprecipitated with an anti-VSV antibody (against IMPα1 or IMPα1IBB) and detected with an anti-V5 antibody. Similar results were obtained in three independent experiments. (B) S2 cells were transiently transfected with pIZ-<i>tim</i>-V5, pAc-<i>per</i>-HA, pIZ-<i>imp_α1</i>-VSV, or pIZ-<i>imp_α1</i>∆IBB-VSV as indicated. After 60 hours, cell lysates were immunoprecipitated with an anti-HA antibody (against PER) and detected with an anti-V5 antibody or with an anti-VSV. Similar results were obtained in three independent experiments. IMPα1 co-immunoprecipitated with PER is indicated with an asterisk. (C) S2 cells were transfected with pIZ-<i>tim</i>-V5 (wt), pIZ-<i>tim</i>^PL-V5, or pIZ-<i>tim</i>^TA-V5 in the presence or absence of pIZ-<i>imp_α1</i>∆IBB-VSV as indicated. After 60 hours, cells were subjected to IP using an anti-VSV antibody (against empty vector or IMPα1∆IBB) and detected with an anti-V5 antibody. Similar results were obtained in three independent experiments. The quantification of interaction between IMPα1 and TIM^WT/PL/TA is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004974#pgen.1004974.s009" target="_blank">S9 Fig.</a></p

Molecular oscillations of PER and TIM are dampened in Df(3L)α1S1 flies.

<p>(A) Quantitive PCR (qPCR) reveals that <i>per</i> and <i>tim</i> mRNA oscillations are blunted in Df(3L)α1S1 flies relative to those in wild-type flies. <i>importin α1</i> mRNA levels do not cycle. <i>actin</i> was used as an internal control to normalize transcript levels. The quantification curves in each panel were plotted as average ± standard error of the mean (SEM) of three independent experiments. (B and C) Western blots of adult fly heads of wild-type (iso³¹), heterozygous, and homozygous Df(3L)α1S1 flies were probed for PER and TIM. Flies of the indicated genotypes were collected (B) at different zeitgeber times (ZT) on the 3^rd day of LD (LD3) and (C) at different circadian times (CT) on the 1^st day of DD (DD1). The blots were also probed with antibody to Hsp70 as a loading control. Similar results were obtained in two or three independent experiments. The quantification of TIM expression levels is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004974#pgen.1004974.s005" target="_blank">S5 Fig.</a></p

NUP153 and RAN are required for circadian rhythms and interact with TIM.

<p>(A) Knockdown of Nup153 disrupts rest:activity rhythms. Representative activity records of individual pdf-Gal4/+; dicer/+ (left) and pdf-Gal4/<i>nup153</i> RNAi; dicer/+ (right) flies are shown. (B) PDF expression in nup153 knockdown flies was detected in l-LNvs, but not in the s-LNvs. In nup153 knockdown flies (right), the s-LNv dorsal tract was impaired as indicated by the white arrow. Low, but cytoplasmic expression of PER (green) was detected in l-LNvs at ZT1. In control flies (left), PER (green) was nuclear in both s- and l-LNvs at ZT1. (C) S2 cells were transfected with pIZ-V5 or pIZ-<i>tim</i>-V5 constructs. After 60 hours, cell lysates were immunoprecipitated with an anti-V5 antibody and western blots were probed with an anti-NUP153 antibody (left). GST pulldown assays were also conducted to test for an interaction between the FG repeat of NUP153 (GST-FG) and TIM (right). Cell extracts from S2 cells transfected with pIZ-<i>tim</i>-V5 were incubated with purified recombinant GST-FG or a GST control. Proteins pulled down by GST were analyzed with an anti-V5 antibody (that recognizes TIM-V5). Similar results were obtained in three independent experiments. (D) Expression of a dominant negative form of Ran (RanDN) in PDF⁺ cells during adulthoods leads to arrhythmia in DD. RanDN was expressed under control of an RU486-inducible <i>Pdf</i>-GS driver. Flies were fed either 500 mM RU486 or ethanol (EtOH, vehicle control) from the time of entrainment. (E) GST pulldown experiments detect interactions between TIM expressed in S2 cells and purified GST-RAN fusion proteins. RAN was tested in GDP-bound (RanGDP) and GTP-bound (RanGTP) forms. GST alone served as a control. The bound protein was analyzed with an anti-V5 antibody. In five independent experiments we have confirmed that TIM shows slight preference (1.64 ± 0.18) for Ran GTP over Ran GDP. (F) Expression of RanDN in central clock cells leads to severe morphological defects (white arrow) on the 3^rd day after RU486 treatment. On the 1^st day after RU486 treatment, morphological effects were not visible and PER (green) was cytoplasmic in both l-LNvs and s-LNvs at ZT1.</p

IMPα1 overexpression increases PER/TIM nuclear translocation in S2 cells.

<p>(A and B) S2 cells were transiently transfected with pCaspeR-<i>per</i>-<i>cfp</i>, pCaspeR-<i>tim</i>-<i>yfp</i>, and pIZ-<i>importin α1</i>-VSV as indicated. (A) Cells were fixed 8 hrs after heat shock induction and then scored as nuclear (blue), cytoplasmic (orange), and uniform (both nuclear and cytoplasmic; gray). At least 100 cells were counted for each condition in two independent experiments. (B) Examples of each condition. The white arrowhead indicates nuclear envelope association of TIM (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004974#sec003" target="_blank">Results</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004974#pgen.1004974.s007" target="_blank">S7B Fig.</a>). (C) S2 cells were transfected as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004974#pgen.1004974.g004" target="_blank">Fig. 4A</a> and then fixed every two hours after heat shock induction. S2 cells were counted as shown above.</p