Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs

An AU rich element (ARE) in the 3' noncoding region promotes the rapid degradation of mammalian cytokine and proto-oncogene mRNAs, such as tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF) and c-fos. Destabilization of ARE-mRNAs involves the association of ARE-binding proteins tristetraprolin or AUF1 and proteasome activity, of which the latter has not been characterized. Here, we show that the stability of a model short-lived mRNA containing the GM-CSF ARE was regulated by the level of ubiquitin-conjugating activity in the cell, which links ARE-mRNA decay to proteasome activity. Increased expression of a cytokine-inducible deubiquitinating protein (DUB) that impairs addition of ubiquitin to proteins fully blocked ARE-mRNA decay, whereas increased expression of a DUB that promotes ubiquitin addition to proteins strongly accelerated ARE-mRNA decay. ARE-mRNA turnover was found to be activated by the ubiquitin-addition reaction and blocked by the ubiquitin-removal reaction. Saturation of the ARE-mRNA decay machinery by high levels of ARE-mRNA, which is well established but not understood, was found to be relieved by increased expression of a DUB that promotes ubiquitin addition to proteins. Finally, inhibition of proteasome activity also blocked accelerated ARE-mRNA decay that is mediated by increased ubiquitin recycling. These results demonstrate that both ubiquitinating activity and proteasome activity are essential for rapid turnover of a model cytokine ARE-mRNA containing the GM-CSF ARE.     

Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8+/-10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9+/-11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1+/-11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3+/-13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE-induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss' myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.     

A comparison of esmolol and diltiazem for heart rate control during coronary revascularisation on beating heart

This prospective study compared control of heart rate and haemodynamics during coronary artery revascularization without cardiopulmonary bypass using either esmolol or diltiazem. Sixty adult patients with one or two vessel coronary artery disease, were randomly divided into 2 groups. Group A (n=30) received a 50 microg/kg/ loading dose of esmolol followed by a 100 microg/kg/hr infusion, for control of heart rate during surgical anastomosis of the coronary vessel. Group-B (n=30) received 0.15 mg/kg of diltiazem as a loading dose followed by a 5 mg/hr infusion for heart rate control, during the anastomosis. It was seen that heart rate control was better in group A, 51.4 +/- 1.3 beats/min, (p <0.01) than in group B, 69.6 +/- 3.0 beats/min (p <0.05), as compared to baseline values of 80.6 +/- 12.1 beats/min in group A and 82.4 +/- 10.6 beats/min in group B respectively. Systemic vascular resistance and pulmonary artery wedge pressure were unchanged in group A but mean pulmonary artery pressure and pulmonary vascular resistance were significantly raised. Group B patients had decreased systemic vascular resistance, mean pulmonary artery pressure and pulmonary artery wedge pressure, and reduced right ventricular stroke work index at the time of distal coronary anastomosis. We concluded that although esmolol provided dramatically slower heart rates, during surgery, the resulting elevations in mean pulmonary artery pressure and pulmonary vascular resistance would require caution if used in patients with underlying right ventricular dysfunction from ischaemia or infarction. Diltiazem by virtue of its effects on systemic vascular resistance, cardiac output, and lowering of mean arterial pressure may be a better choice in hypertensive patients.     

Perforator Peroneal Artery Flap for Tongue Reconstruction

Introduction

Reconstruction has evolved long way from primary closure to flaps. As time evolved, better understanding of vascularity of flap has led to the development of innovative reconstructive techniques. These flaps can be raised from various parts of the body for reconstruction and have shown least donor site morbidity. We use one such peroneal artery perforator flap for tongue reconstruction with advantage of thin pliable flap, minimal donor site morbidity and hidden scar.

Materials and Methods

Our patient 57yrs old lady underwent wide local excision with selective neck dissection. Perforators are marked about 10 and 15 cm inferiorly from the fibular head using hand held Doppler. Leg is positioned in such a way to give better exposure during dissection of the flap and flap is harvested under a tourniquet with pressure kept 350 mm Hg. The perforator is kept at the eccentric location, so as to gain length of the pedicle. Skin incison is placed over the peroneal muscle and deepened unto the deep facia, then the dissection is continued over the muscle and the perforator arising from the lateral septum. The proximal perforator about 10 cm from the fibular head is a constant perforator and bigger one, which is traced up to the peroneal vessel. We could get a 6 cm of pedicle length. Finally the flap is islanded on this perforator and the pedicle is ligated and flap harvested. Anastamosis was done to the ipsilateral side to facial vessels. The donor site is closed primarily and in the upper half one can harvest 5 cm width flap without requiring a skin graft along with a length of 8 to 12 cm.

Discussion

Various local and free flap has been used for reconstruction of partial tongue defects with its obvious donor site problems, like less pliable skin and not so adequate tissue from local flaps and sacrificing a important artery as in radial forearm flap serves as the work horse in reconstruction of partial tongue defects, Concept of super microsurgery was popularized by Japanese in 1980s and the concept of angiosome proposed by Taylor paved the way for development of new flaps. True perforator flaps are those where the source vessel is left undisturbed and overlying skin flap is raised. Yoshimura proposed cutaneous flap could be raised from peroneal artery (Br J Plast Surg 42:715–718, 1989). Wolff et al. (Plast Reconstr Surg 113:107–113, 2004) first used perforator based peroneal artery flap for oral reconstruction. Location of perforators vary, hence pre operative localisation can be done by ultrasound doppler, CT angio or MR angiography. Disadvantages over radial flap include varying anatomic location of perforators, need for imaging and difficult dissection of delicate vessels through muscles and hence a learning curve. Our patient had an arterial thrombus within few hours post-operatively which was successfully salvaged with immediate re-exploration and re-anastomosis of artery. Post-operative healing was uneventful and donor site was closed primarily without the need for graft.

Conclusions

Perforator peroneal flap serves as a useful armamentarium for reconstruction of moderate size defects of tongue, buccal mucosa and floor of mouth with advantages of thin pliable flap, minimal donor site morbidity and hidden scar.

     

Large-area formation of self-aligned crystalline domains of organic semiconductors on transistor channels using CONNECT

The electronic properties of solution-processable small-molecule organic semiconductors (OSCs) have rapidly improved in recent years, rendering them highly promising for various low-cost large-area electronic applications. However, practical applications of organic electronics require patterned and precisely registered OSC films within the transistor channel region with uniform electrical properties over a large area, a task that remains a significant challenge. Here, we present a technique termed “controlled OSC nucleation and extension for circuits” (CONNECT), which uses differential surface energy and solution shearing to simultaneously generate patterned and precisely registered OSC thin films within the channel region and with aligned crystalline domains, resulting in low device-to-device variability. We have fabricated transistor density as high as 840 dpi, with a yield of 99%. We have successfully built various logic gates and a 2-bit half-adder circuit, demonstrating the practical applicability of our technique for large-scale circuit fabrication.Organic electronics are being rapidly developed to pave the way for low-cost, large-area, flexible, and transparent electronics, such as active matrix displays, radiofrequency identification tags, and integrated logic circuits (1–6). Solution-processable small-molecular organic semiconductors (OSCs) are promising for these applications because of their high performance in organic thin-film transistors (OTFTs) and their low-cost, large-area processability (7–11). Recently, solution-processed small-molecule OSCs have shown record-breaking charge carrier mobility in thin-film transistors, furthermore providing the exciting prospect of organic electronics (9–12). However, significant challenges still remain to realize practical applications of organic electronics. First, patterning and precisely registering OSCs within the channel region is imperative to reduce leakage current between neighboring TFTs and parasitic capacitance, and to increase device yield. Second, low variability in electrical characteristics is essential so that all OTFTs work in concert with one another to carry out proper device functionalities. Last, all these features must be achieved in a scalable and economical manner, as large area and low cost are key advantages for organic electronics.To address the above challenges, various groups have developed methods to pattern OSCs (3, 9, 13–24), such as templating OSCs (14, 19–21), inkjet printing (9, 18), gravure printing, and other roll-to-roll coating methods (25–27). In addition, the use of solvent wetting/dewetting surface treatments has been widely used in conjunction with the patterning methods described above (10, 11, 13, 15–17, 22, 28, 29). Despite some promising results, there remain shortcomings that potentially limit their practical applicability. Primarily, it is difficult to align drain and source electrodes to OSCs, as standard photolithography generally cannot be directly applied due to solvent-induced degradation. Additive printing methods to place OSCs on electrodes or vice versa is often difficult to achieve, especially as the density of TFTs become higher (30). For instance, alignment using inkjet printing is restricted by the registration capability of the instrument, whereas the size of each OTFT is limited by the droplet size attainable (22, 31). Another potential issue with these methods is the variance in electrical characteristics of TFTs due to the difficulty in obtaining uniform crystal growth on isolated TFTs over large areas (9, 15, 21, 27, 28, 31–35). Even single crystalline domains of patterned TFTs may have large variability in electrical characteristics as a result of random orientation and size variance.Self-alignment of OSCs on electrodes has been developed by various groups to overcome misalignment (36, 37). Vertical TFTs were created to achieve self-aligned small channels (36, 37); however, achieving high-device performance requires precise control of device fabrication. UV light exposure after electrode patterning can be used to generate a hydrophilic channel between the electrodes, where OSCs can be selectively isolated and deposited (38–40). However, this method is limited to transparent substrates and only short channels (<500 nm). The UV light can also damage the OSC–dielectric interface, creating traps that increase the variability between TFTs. Contact directed nucleation have previously been used to increase crystallinity in the channel region (41). However, grain boundaries remain and result in large variance in OTFT performance. Mannsfeld et al. (42) have reported solution deposition of OSC crystals on electrodes using a liquid vortex method, but the OSC crystals have incomplete channel coverage, leading to varying electrical characteristics. Polycrystalline thin films of pentacene have been formed in between electrodes using vapor deposition, but patterning is required to isolate the area of deposition (43).In this study, we introduce a highly effective method—controlled OSC nucleation and extension for circuits (CONNECT)—that uses differential surface wetting properties combined with solution shearing (44, 45) to pattern and align crystalline domains of OSCs selectively between source and drain electrodes in a single step. This method of patterning overcomes a number of challenges previously encountered with other patterning methods described above. First, as the electrodes themselves serve as templates for patterning OSCs, extra steps to pattern and register OSCs to the channel region are not necessary. Second, this method ensures that each bridging event yields aligned crystalline domains from a single nucleation site across the channel, which results in OTFTs with relatively low variance in electrical characteristics. Our method was observed to work over a wide range of channel lengths, ranging from 0.5 to 20 µm, both with photolithographically patterned and inkjet-printed electrodes, making our process versatile to different electrode types and a wide range of OTFT densities. We have attained OTFT densities as high as 840 dpi, which is among the highest achieved in OTFTs (46). Last, our method was shown to be scalable to a large area and had a high crystal CONNECT yield of 99%. As a first demonstration of the potential of our method, we have fabricated various logic gates and a 2-bit half-adder circuit consisting of 60 source/drain channel regions. Together, these demonstrations render our method highly promising for various organic electronic applications where ease of processability, scalability, flexibility in OTFT density, device-to-device uniformity, and yield are of great importance.     

Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5–Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.Erythrocyte invasion by Plasmodium falciparum merozoites is crucial for malaria pathogenesis, and thus the parasite has evolved an extensive molecular machinery to ensure invasion through multiple pathways (1–3). The quest to develop successful blood-stage malaria vaccines that efficiently block this process have focused on essential parasite proteins like merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1); however, these are highly polymorphic, unable to elicit strain-transcending neutralizing antibodies, and have thus failed in field trials (4). Among the large repertoire of invasion-related proteins, the family of P. falciparum reticulocyte binding-like homologous (PfRH) proteins have emerged as key determinants of different invasion pathways (2, 3), of which PfRH5 is the only essential conserved parasite ligand (5–8) that elicits potent strain-transcending neutralizing antibodies (9–12). It is localized in the rhoptry and secreted to the merozoite surface during erythrocyte invasion (6). It does not seem to be under immune pressure (9, 13) and is favored to be a leading vaccine candidate. PfRH5 has been shown to interact with another parasite protein, PfRipr (P. falciparum RH5 interacting protein) (14). However, both these proteins lack transmembrane domains as well as a GPI anchor, and thus the mechanism through which PfRH5 is secured on the surface of an invading merozoite to facilitate its functional role during invasion still remains unknown. It is likely that PfRH5 might be attached to the merozoite surface as a complex with other essential proteins other than PfRipr, identification of which could open new therapeutic avenues against malaria.Here we show that PfRH5 and PfRipr interact with a GPI-linked parasite protein, CyRPA (Cysteine-rich protective antigen) (15) to form an essential complex on the surface of an invading merozoite. Individual antibodies against each of the three proteins successfully coimmunoprecipitated all three proteins, confirming their presence as a multiprotein complex. Analysis of the native parasite protein complex by different chromatographic techniques further confirmed that all three protein components coeluted together and were present as a much higher molecular mass species than their individual molecular masses. We also demonstrated that the three proteins are colocalized on the apical surface of the invading merozoite, of which only CyRPA was shown to be GPI-linked. Importantly, antibodies against full-length CyRPA potently blocked erythrocyte invasion by multiple P. falciparum strains, as observed previously only for PfRH5 antibodies (9–12). Because CyRPA does not bind with the erythrocyte surface, it seems that the parasite-neutralizing CyRPA antibodies function by impeding its interaction with PfRH5 or PfRipr. Hence, we have identified and validated a GPI-linked parasite protein, CyRPA, as another essential interacting partner of PfRH5 that is responsible for tethering it to the merozoite surface. Further, we have shown that like PfRH5, CyRPA is a conserved target of potent antibody-mediated blockade of erythrocyte invasion and thus seems to be another highly promising blood-stage vaccine candidate.     

Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2A^Cdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle.The eukaryotic cell cycle is a highly coordinated and conserved process. In addition to DNA replication, one of the major requirements for the cell to progress through the cell cycle is the precise duplication of membrane-enclosed organelles and other cellular components before cell division. Knowledge about the mechanisms regulating (membrane) lipid homeostasis during the cell cycle is scarce (1), however several levels of evidence suggest regulation of key enzymes of lipid metabolism in a cell-cycle–dependent manner. The PAH1-encoded phosphatidic acid (PA) phosphatase (Pah1), a key enzyme of triacylglycerol (TG) synthesis that provides the TG precursor diacylglycerol (DG), is phosphorylated and inactivated by the cyclin-dependent kinases Cdc28 and Pho85–Pho80 (2, 3). Kurat et al. showed that Tgl4, next to Tgl3, one of the two major TG lipases in yeast and the ortholog of mammalian ATGL (4, 5), is also phosphorylated by Cdc28. In contrast to Pah1, however, Tgl4 is activated by Cdc28 (6). This inverse regulation of Pah1 and Tgl4 by Cdc28-dependent phosphorylation led to the model by which the TG content oscillates during the cell cycle: On the one hand, TG synthesis serves as a buffer for excess de novo generated fatty acids (FAs), and on the other hand, in times of increased demand—that is, at the onset of bud formation and bud growth—Tgl4-catalyzed lipolysis becomes active to provide TG-derived precursors for membrane lipid synthesis (6).TG and membrane phospholipids share the same intermediates, PA and DG; PA is generated by sequential acylation of glycerol-3-phosphate, reactions that mostly take place in the endoplasmic reticulum (ER) membrane (7). The dephosphorylation of PA to DG by the PAH1-encoded PA phosphatase Pah1 is the major regulator of cellular TG synthesis in yeast (8), similar to its mammalian ortholog, lipin (9). According to this central role, TG content in PAH1-deficient yeast cells is decreased by 70–90%; the source of the residual TG in these mutants is currently unclear. TG synthesis from DG requires one additional acylation step that is catalyzed by the DGA1-encoded acyl-CoA–dependent DG acyltransferase (10, 11) and the phospholipid-dependent acyltransferase, encoded by LRO1 (7). Alternatively, in the presence of the phospholipid precursors, ethanolamine and/or choline, DG may be converted into phospholipids via the Kennedy pathway (7). Thus, net TG synthesis in growing cells is determined by multiple factors, including the availability of FAs, presence of lipid precursors, and the activities of PA phosphatase and the DG acyltransferases. Degradation of TG in yeast is governed by the major lipid droplet (LD)-associated lipases, encoded by TGL3 and TGL4 (4, 12); both enzymes belong to the patatin-domain–containing family of proteins, members of which play a crucial role in lipid homeostasis also in mammals (13). Multiple additional lipases exist in yeast, but their specific function and contribution to TG homeostasis may be restricted to specific growth conditions (7, 14, 15).Absence of lipolysis in mutants lacking TGL3 and TGL4 results in up to threefold elevated levels of TG and reduced levels of phosphatidylcholine and sphingolipids (4, 12, 16, 17), indicating that TG breakdown provides precursors for these lipids or generates some regulatory factors required for their synthesis. The rate of phosphatidylinositol (PI) synthesis after readdition of inositol to inositol-starved cells is reduced by 50% in lipase-deficient cells; the boost of PI synthesis under inositol refeeding conditions is completely abolished if de novo FA synthesis is additionally blocked in the lipase mutants by the inhibitor cerulenin (18). These data clearly demonstrate the requirements for TG breakdown, in addition to de novo FA synthesis, to generate precursors for membrane lipids. As a consequence of defective lipolysis, entry of quiescent cells into vegetative growth is significantly delayed; thus, TG breakdown is particularly important for promoting exit from the stationary phase and entry into the gap1 (G1) phase of the cell cycle (4, 6, 19).Progression through the cell cycle is regulated by specific checkpoint pathways that ensure completion of crucial events and execute a halt under nonconducive conditions. Checkpoint mechanisms slow down or arrest the cell cycle to enable cells to fix damage or to obtain the required metabolites before proceeding and are as such important for the integrity of cell division (20–22). According to this critical function in quality control, mutations in checkpoint genes in mammals have been linked to cancer predisposition and progression. The first discovered cell-cycle checkpoint in Schizosaccharomyces pombe that regulates entry into mitosis is executed by the Wee1 kinase (23, 24), which delays mitosis by phosphorylating and inhibiting cyclin-dependent kinase Cdk1 (25). Conversely, the phosphatase Cdc25 promotes entry into mitosis by removing the inhibitory phosphorylation of Cdk1 (26–28). The budding yeast orthologs of Wee1 and Cdc25 are called Swe1 and Mih1, and their key functions in regulating Cdk1 activity are highly conserved (29, 30). Swe1 phosphorylates Cdk1 (encoded by CDC28 in budding yeast) at the tyrosine 19 residue and inhibits its kinase activity (29, 31, 32); the Mih1 phosphatase removes this inhibitory phosphorylation initiating G2/M cell-cycle progression (26). The Swe1 and Cdk1/Cdc28 kinases operate in an autoregulatory loop in which Swe1 is initially phosphorylated and activated by Cdk1/Cdc28 that is associated with mitotic cyclins; subsequently, activated Swe1 phosphorylates and inhibits Cdk1/Cdc28 (33). The initial phosphorylation of Swe1 is opposed by the protein phosphatase 2A (PP2A) with its catalytic subunits Pph21 or Pph22 and the regulatory subunit Cdc55 (PP2A^Cdc55), which sets a threshold, limiting the activation of Swe1 by Cdk1/Cdc28 in early mitosis (34, 35). Loss of the regulatory subunit Cdc55 leads to hyperactivation of Swe1 (35); after the initial phosphorylation of Swe1 in early mitosis, subsequent phosphorylation events trigger full hyperphosphorylation of Swe1 (33), which leads to its ubiquitin-mediated degradation (36, 37). Of note, regulation of Cdk1/Cdc28 by the G1 cyclin Cln2 plays an important role in actin cytoskeleton polarization and the localized delivery of secretory vesicles, which contribute membrane lipids to the developing bud, thus linking cell surface growth to the cell cycle (38).Despite its proposed role as a gap2 phase (G2) checkpoint regulator, we now show that Swe1 kinase is responsible for the G1/S (Gap1/replication phase) cell-cycle delay in mutants defective in TG lipolysis by phosphorylating Cdk1/Cdc28 at tyrosine 19. Deletion of Swe1 in the tgl3 tgl4 lipase mutant restores normal cell-cycle progression; similarly, supplementation of mutant cells with saturated FAs (myristic acid, palmitic acid) or a precursor of sphingolipid synthesis, phytosphingosine (PHS), suppress the cell-cycle delay in the lipase mutants. These data suggest that Swe1 is a lipid-regulated kinase that is activated in the absence of specific lipids, presumably sphingolipids, and halts G1/S transition by phosphorylating Cdk1/Cdc28 in lipase-deficient cells that exit from the G0 phase of the cell cycle.