Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Vesicular Stomatitis Virus Induces Apoptosis Primarily through Bak Rather than Bax by Inactivating Mcl-1 and Bcl-XL

Vesicular stomatitis virus (VSV) induces apoptosis via the mitochondrial pathway. The mitochondrial pathway is regulated by the Bcl-2 family of proteins, which consists of both pro- and antiapoptotic members. To determine the relative importance of the multidomain proapoptotic Bcl-2 family members Bak and Bax, HeLa cells were transfected with Bak and/or Bax small interfering RNA (siRNA) and subsequently infected with recombinant wild-type VSV. Our results showed that Bak is more important than Bax for the induction of apoptosis in this system. Bak is regulated by two antiapoptotic Bcl-2 proteins, Mcl-1, which is rapidly turned over, and Bcl-X_L, which is relatively stable. Inhibition of host gene expression by the VSV M protein resulted in the degradation of Mcl-1 but not Bcl-X_L. However, inactivation of both Mcl-1 and Bcl-X_L was required for cells to undergo apoptosis. While inactivation of Mcl-1 was due to inhibition of its expression, inactivation of Bcl-X_L indicates a role for one or more BH3-only Bcl-2 family members. VSV-induced apoptosis was inhibited by transfection with siRNA against Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, the initiator caspase associated with the death receptor pathway. Similarly, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.The induction of cell death is a major mechanism by which many viruses cause disease in the tissues they infect (23). In addition, the cytolytic activity of viruses has the potential for therapeutic applications, such as the development of oncolytic viruses for the treatment of cancer (27). Vesicular stomatitis virus (VSV) is well studied as a prototype for negative-strand RNA viruses and is an exceptionally potent inducer of apoptosis in a wide variety of cell types (4, 20, 21). Due to its particularly rapid cytopathic effects, VSV is one of the major viruses being developed as an oncolytic agent (27). VSV is capable of inducing apoptosis by activation of multiple apoptotic pathways. It is important to determine how these pathways are activated and the role that they play in apoptosis induced by VSV in order to understand the virulence and oncolytic activity of the virus, as well as to provide a model to which other viruses can be compared.Previous work showed that wild-type (wt) VSV induces apoptosis via the mitochondrial (intrinsic) pathway through the initiator caspase caspase-9 (4, 19). This is due in part to the inhibition of host gene expression by the VSV M protein (19). The inhibition of host gene expression by M protein is the mechanism by which VSV inhibits the host antiviral response (2, 31) and leads to induction of apoptosis, similar to that induced by pharmacologic inhibitors of host gene expression (19). Additionally, M protein mutants of VSV that are deficient in the ability to inhibit new host gene expression are effective inducers of apoptosis (12, 13, 19, 20). However, in contrast to wt VSV, induction of apoptosis by M protein mutant virus occurs primarily via the extrinsic pathway through the initiator caspase caspase-8 (12, 13). Infection with M protein mutant VSV results in the expression of proapoptotic genes that are suppressed during infection with wt VSV (12). Therefore, in the case of VSV with wt M protein, the induction of apoptosis is most likely mediated by proteins already present in the host cell. Since it has previously been shown that wt VSV activates the intrinsic pathway, we focused on the Bcl-2 family of proteins to determine the role of Bcl-2 family members in apoptosis induced by wt VSV.Bcl-2 family proteins function to either suppress or promote mitochondrial outer membrane permeabilization, thereby regulating the release of proapoptotic factors into the cytosol, such as cytochrome c, apoptosis-inducing factor (AIF), and Smac/Diablo (5). Bcl-2 family proteins are subdivided into three groups, depending on the conservation of Bcl-2 homology (BH) domains and function (reviewed in references 8 and 38). The multidomain antiapoptotic Bcl-2 proteins contain BH domains BH1 to BH4 and function to inhibit apoptosis by binding to proapoptotic Bcl-2 family members. Members of this group include Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and BFL-1/A1. The proapoptotic Bcl-2 proteins are comprised of two groups, the multidomain proteins and the BH3-only proteins. Bax and Bak are the two main members of the multidomain group, containing BH domains BH1 to BH3. These proteins are primarily responsible for the permeabilization of the mitochondrial outer membrane, if their activity is not suppressed by antiapoptotic Bcl-2 family members. The BH3-only proteins contain only one Bcl-2 homology domain (BH3) and include Bid, Bad, Bim, Puma, Noxa, and Bik, among others. These proteins function as upstream sensors of signaling pathways and convey to other Bcl-2 family proteins the signals to initiate apoptosis. These death signals can be transmitted from the BH3-only proteins by either binding to antiapoptotic proteins, causing the release of Bak and Bax, or binding to Bak and Bax, thereby causing their activation (6).The pathways leading to activation of Bak differ from those that activate Bax. Interestingly, only two antiapoptotic Bcl-2 proteins, Mcl-1 and Bcl-X_L, have been shown to interact with Bak, while Bax appears to be able to interact with all of the antiapoptotic proteins, with the exception of Mcl-1 (7, 35). BH3-only proteins have strong binding affinities to the antiapoptotic proteins, suggesting that their primary role may be to derepress Bak and Bax by binding and inhibiting the antiapoptotic proteins (36). In addition, BH3-only proteins may play a role in activation of Bak and Bax by binding and inducing an activated conformation (6, 34). For some stimuli, such as the protein kinase inhibitor staurosporine (SSP), the topoisomerase II inhibitor etoposide, and UV radiation, Bak and Bax appear to be redundant, in that the deletion of both is required to render cells resistant to these agents (33). In contrast, Bak and Bax were nonredundant in the induction of apoptosis by Neisseria gonorrhoeae and cisplatin, such that both were required for apoptosis to occur (18).In the experiments reported here, the silencing of Bak or Bax expression with small interfering RNA (siRNA) showed that Bak is more important than Bax for the induction of apoptosis in HeLa cells infected with wt VSV. Overexpression of both of the antiapoptotic Bcl-2 family proteins known to interact with Bak, Mcl-1 and Bcl-X_L, delayed the onset of apoptosis, while depletion of Mcl-1 or Bcl-X_L by siRNA transfection prior to infection increased the rate of apoptosis. Furthermore, M protein inhibition of new host gene expression led to the depletion of Mcl-1, enabling the rapid activation of apoptosis. However, inhibition of Bcl-X_L was also required for the initiation of apoptosis, indicating a role for one or more BH3-only proteins. Bid, a BH3-only protein that is normally activated by the cleavage of caspase-8, was shown to be important for induction of apoptosis by VSV. Likewise, treatment with an inhibitor of caspase-8 inhibited VSV-induced apoptosis. These results indicate a role for cross talk from the death receptor pathway in the activation of the mitochondrial pathway by VSV.     

Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors

The cellular pathways of apoptosis have not been fully characterized; however, calpain, a cytosolic calcium-activated cysteine protease, has been implicated in several forms of programmed cell death. Reoviruses induce apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of calpain in reovirus-induced apoptosis in vitro by measuring calpain activity as well as evaluating the effects of calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and calpain activity, measured as cleavage of the fluorogenic calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of apoptosis by calpain inhibitors is independent of effects on viral replication. We conclude that calpain activation is a component of the regulatory cascade in reovirus-induced apoptosis.     

Accumulation of the pro-apoptotic factor Bak is controlled by antagonist factor Mcl-1 availability

Apoptosis has become recognized as a crucial mechanism involved in a wide range of physiological and pathological processes. Following an initial pro-apoptotic signal, controlling phases allow the cell to reinforce or downgrade signals leading to the irrevocable entry into apoptosis. Bak (Bcl-2-antagonist killer) is a mitochondrial pore-forming pro-apoptotic effector inhibited through titration by the anti-apoptotic protein Mcl-1 (Myeloid cell leukemia-1). Viruses have taken advantage of proteasome-dependent degradation of Bak as a mechanism to prevent apoptosis in infected cells. It is not clear however whether regulation of Bak protein level is involved in other physiological processes. In this report, we show that Mcl-1 level is paralleled by Bak while a Mcl-1 non-interacting mutant of Bak does not accumulate in cells. This mechanism is proteasome independent. Following serum withdrawal, Bak accumulation becomes independent of Mcl-1 level and cells are sensitized to pro-apoptotic stimuli. Based on these results, we propose that regulation of Mcl-1-Bak steochiometry is a control mechanism used as a checkpoint to prevent or allow entry into apoptosis.     

Proteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-737 in Melanoma Cells

Background and purpose

Metastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.

Experimental approach

For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.

Key results

The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC₅₀ of between 2.2–5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.

Conclusions and implications

Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.     

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Apoptosis Induced by Ginkgo biloba (EGb761) in Melanoma Cells Is Mcl-1-Dependent

Melanoma is an aggressive skin cancer. Unfortunately, there is currently no chemotherapeutic agent available to significantly prolong the survival of the most patients with metastatic melanomas. Here we report that the Ginkgo biloba extract (EGb761), one of the most widely sold herbal supplements in the world, potently induces apoptosis in human melanoma cells by disturbing the balance between pro- and anti-apoptosis Bcl-2 family proteins. Treatment with EGb761 induced varying degrees of apoptosis in melanoma cell lines but not in melanocytes. Induction of apoptosis was caspase-dependent and appeared to be mediated by the mitochondrial pathway, in that it was associated with reduction in mitochondrial membrane potential and activation of Bax and Bak. Although EGb761 did not cause significant change in the expression levels of the BH3-only Bcl-2 family proteins Bim, Puma, Noxa, and Bad, it significantly downregulated Mcl-1 in sensitive but not resistant melanoma cells, suggesting a major role of Mcl-1 in regulating apoptosis of melanoma cells induced by EGb761. Indeed, siRNA knockdown of Mcl-1 enhanced EGb761-induced apoptosis, which was associated with increased activation of Bax and Bak. Taken together, these results demonstrate that EGb761 kills melanoma cells through the mitochondrial apoptotic pathway, and that Mcl-1 is a major regulator of sensitivity of melanoma cells to apoptosis induced by EGb761. Therefore, EGb761 with or without in combination with targeting Mcl-1 may be a useful strategy in the treatment of melanoma.     

Differential regulation of Bax and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1

The pro-apoptotic members of the Bcl-2 family include initiator proteins that contain only BH3 domains and downstream effector multi-BH domain-containing proteins, including Bax and Bak. In this report, we compared the ability of the six human anti-apoptotic Bcl-2 family members to suppress apoptosis induced by overexpression of Bax or Bak, correlating findings with protein interactions measured by three different methods: co-immunoprecipitation, glutathione S-transferase pulldown, and fluorescence polarization assays employing synthetic BH3 peptides from Bax and Bak. Bcl-B and Mcl-1 showed strong preferences for binding to and suppression of Bax and Bak, respectively. In contrast, the other anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-X(L), Bcl-W, and Bfl-1) suppressed apoptosis induced by overexpression of either Bax or Bak, and they displayed an ability to bind both Bax and Bak by at least one of the three protein interaction methods. Interestingly, however, full-length Bax and Bak proteins and synthetic Bax and Bak BH3 peptides exhibited discernible differences in their interactions with some anti-apoptotic members of the Bcl-2 family, cautioning against reliance on a single method for detecting protein interactions of functional significance. Altogether, the findings reveal striking distinctions in the behaviors of Bcl-B and Mcl-1 relative to the other anti-apoptotic Bcl-2 family members, where Bcl-B and Mcl-1 display reciprocal abilities to bind and neutralize Bax and Bak.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

The voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H⁺-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H⁺ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pH_I). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis.     

UMI-77 primes glioma cells for TRAIL-induced apoptosis by unsequestering Bim and Bak from Mcl-1

Nucleotidases participate in the regulation of physiological and pathological events, such as inflammation and coagulation. Exercise promotes distinct adaptations, and can influence purinergic signaling. In the present study, we investigated soluble nucleotidase activities in the blood serum of sedentary young male adults at pre- and post-acute moderate aerobic exercise. In addition, we evaluated how this kind of exercise could influence adenine nucleotide concentrations in the blood serum. Sedentary individuals were submitted to moderate aerobic exercise on a treadmill; blood samples were collected pre- and post-exercise, and serum was separated for analysis. Results showed increases in ATP, ADP, and AMP hydrolysis post-exercise, compared to pre-exercise values. The ecto-nucleotide pyrophosphatase/phosphodiesterase was also evaluated, showing an increased activity post-exercise, compared to pre-exercise. Purine levels were analyzed by HPLC in the blood serum, pre- and post-exercise. Decreased levels of ATP and ADP were found post-exercise, in contrast with pre-exercise values. Conversely, post-exercise levels of adenosine and inosine increased compared to pre-exercise levels. Our results indicate an influence of acute exercise on ATP metabolism, modifying enzymatic behavior to promote a protective biological environment.     

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.     

Nicotine induces cell survival and chemoresistance by stimulating Mcl-1 phosphorylation and its interaction with Bak in lung cancer

Nicotine is a major carcinogen in cigarettes, which can enhance cell proliferation and metastasis and increase the chemoresistance of cancer cells. Our previous data found that nicotine promotes cell survival in lung cancer by affecting the expression of antiapoptotic protein Mcl-1, suggesting that the Mcl-1 may be a therapeutic target for patients with lung cancer. In this study, we found that the effects of drug resistance on nicotine-induced lung cancer cell lines were shown to influence the phosphorylation of Mcl-1. Moreover, nicotine induces Mcl-1 phosphorylation exclusively at the T163 site, which results in enhancement of the antiapoptotic activity of Mcl-1 and increased cell survival. Meanwhile, nicotine can reduce the sensitivity of H1299 cells to CDDP via enhancement of the binding of Mcl-1 to Bak, which inhibits the proapoptotic effect of Bak and ultimately leads to increased survival and drug resistance of lung cancer cells. Thus, nicotine-induced cell survival and chemoresistance may occur in a mechanism by stimulating Mcl-1 phosphorylation and its interaction with Bak, which may contribute to improving the efficacy of chemotherapy in the treatment of human lung cancer.     

M. leprae inhibits apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 gene expression

Background  

Virulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG.     

Glucose Starvation-Induced Dispersal of Pseudomonas aeruginosa Biofilms Is cAMP and Energy Dependent

Carbon starvation has been shown to induce a massive dispersal event in biofilms of the opportunistic pathogen Pseudomonas aeruginosa; however, the molecular pathways controlling this dispersal response remain unknown. We quantified changes in the proteome of P. aeruginosa PAO1 biofilm and planktonic cells during glucose starvation by differential peptide-fingerprint mass-spectrometry (iTRAQ). In addition, we monitored dispersal photometrically, as a decrease in turbidity/opacity of biofilms pre-grown and starved in continuous flow-cells, in order to evaluate treatments (e.g. inhibitors CCCP, arsenate, chloramphenicol, L-serine hydroxamate) and key mutants altered in biofilm development and dispersal (e.g. nirS, vfr, bdlA, rpoS, lasRrhlR, Pf4-bacteriophage and cyaA). In wild-type biofilms, dispersal started within five minutes of glucose starvation, was maximal after 2 h, and up to 60% of the original biomass had dispersed after 24 h of starvation. The changes in protein synthesis were generally not more than two fold and indicated that more than 100 proteins belonging to various classes, including carbon and energy metabolism, stress adaptation, and motility, were differentially expressed. For the different treatments, only the proton-ionophore CCCP or arsenate, an inhibitor of ATP synthesis, prevented dispersal of the biofilms. For the different mutants tested, only cyaA, the synthase of the intracellular second messenger cAMP, failed to disperse; complementation of the cyaA mutation restored the wild-type phenotype. Hence, the pathway for carbon starvation-induced biofilm dispersal in P. aeruginosa PAO1 involves ATP production via direct ATP synthesis and proton-motive force dependent step(s) and is mediated through cAMP, which is likely to control the activity of proteins involved in remodeling biofilm cells in preparation for planktonic survival.