Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks.

Signal transduction pathways activated by chemoattractants have been extensively studied, but little is known about the events mediating responses to mechanical stimuli. We discovered that acute mechanical perturbation of cells triggered transient activation of all tested components of the chemotactic signal transduction network, as well as actin polymerization. Similarly to chemoattractants, the shear flow-induced signal transduction events displayed features of excitability, including the ability to mount a full response irrespective of the length of the stimulation and a refractory period that is shared with that generated by chemoattractants. Loss of G protein subunits, inhibition of multiple signal transduction events, or disruption of calcium signaling attenuated the response to acute mechanical stimulation. Unlike the response to chemoattractants, an intact actin cytoskeleton was essential for reacting to mechanical perturbation. These results taken together suggest that chemotactic and mechanical stimuli trigger activation of a common signal transduction network that integrates external cues to regulate cytoskeletal activity and drive cell migration.

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes.

Chemotaxis, or directed migration of cells along a chemical gradient, is a highly coordinated process that involves gradient sensing, motility, and polarity. Most of our understanding of chemotaxis comes from studies of cells undergoing amoeboid-type migration, in particular the social amoeba Dictyostelium discoideum and leukocytes. In these amoeboid cells the molecular events leading to directed migration can be conceptually divided into four interacting networks: receptor/G protein, signal transduction, cytoskeleton, and polarity. The signal transduction network occupies a central position in this scheme as it receives direct input from the receptor/G protein network, as well as feedback from the cytoskeletal and polarity networks. Multiple overlapping modules within the signal transduction network transmit the signals to the actin cytoskeleton network leading to biased pseudopod protrusion in the direction of the gradient. The overall architecture of the networks, as well as the individual signaling modules, is remarkably conserved between Dictyostelium and mammalian leukocytes, and the similarities and differences between the two systems are the subject of this review.

Tumor suppressor Hippo/MST1 kinase mediates chemotaxis by regulating spreading and adhesion.

Chemotaxis depends on a network of parallel pathways that coordinate cytoskeletal events to bias cell movement along a chemoattractant gradient. Using a forward genetic screen in Dictyostelium discoideum, we identified the Ste20 kinase KrsB, a homolog of tumor suppressors Hippo and MST1/2, as a negative regulator of cell spreading and substrate attachment. The excessive adhesion of krsB(-) cells reduced directional movement and prolonged the streaming phase of multicellular aggregation. These phenotypes depended on an intact kinase domain and phosphorylation of a conserved threonine (T176) within the activation loop. Chemoattractants triggered a rapid, transient autophosphorylation of T176 in a heterotrimeric G protein-dependent and PI3K- and TorC2-independent manner. The active phosphorylated form of KrsB acts to decrease adhesion to the substrate. Taken together these studies suggest that cycling between active and inactive forms of KrsB may provide the dynamic regulation of cell adhesion needed for proper cell migration and chemotaxis. KrsB interacts genetically with another D. discoideum Hippo/MST homolog, KrsA, but the two genes are not functionally redundant. These studies show that Hippo/MST proteins, like the tumor suppressor PTEN and oncogenes Ras and PI3K, play a key role in cell morphological events in addition to their role in regulating cell growth.

Differential Roles of Actin Crosslinking Proteins Filamin and α-Actinin in Shear Flow-Induced Migration of Dictyostelium discoideum.

Shear flow-induced migration is an important physiological phenomenon experienced by multiple cell types, including leukocytes and cancer cells. However, molecular mechanisms by which cells sense and directionally migrate in response to mechanical perturbation are not well understood. Dictyostelium discoideum social amoeba, a well-established model for studying amoeboid-type migration, also exhibits directional motility when exposed to shear flow, and this behavior is preceded by rapid and transient activation of the same signal transduction network that is activated by chemoattractants. The initial response, which can also be observed following brief 2 s stimulation with shear flow, requires an intact actin cytoskeleton; however, what aspect of the cytoskeletal network is responsible for sensing and/or transmitting the signal is unclear. We investigated the role of actin crosslinkers filamin and α-actinin by analyzing initial shear flow-stimulated responses in cells with or without these proteins. Both filamin and α-actinin showed rapid and transient relocalization from the cytosol to the cortex following shear flow stimulation. Using spatiotemporal analysis of Ras GTPase activation as a readout of signal transduction network activity, we demonstrated that lack of α-actinin did not reduce, and, in fact, slightly improved the response to acute mechanical stimulation compared to cells expressing α-actinin. In contrast, shear flow-induced Ras activation was significantly more robust in filamin-null cells rescued with filamin compared to cells expressing empty vector. Reduced responsiveness appeared to be specific to mechanical stimuli and was not due to a change in the basal activity since response to global stimulation with a chemoattractant and random migration was comparable between cells with or without filamin. Finally, while filamin-null cells rescued with filamin efficiently migrated upstream when presented with continuous flow, cells lacking filamin were defective in directional migration. Overall, our study suggests that filamin, but not α-actinin, is involved in sensing and/or transmitting mechanical stimuli that drive directed migration; however, other components of the actin cytoskeleton likely also contribute to the initial response since filamin-null cells were still able to activate the signal transduction network. These findings could have implications for our fundamental understanding of shear flow-induced migration of leukocytes, cancer cells and other amoeboid-type cells.

Catalytically inactive SHIP2 inhibits proliferation by attenuating PDGF signaling in 3T3-L1 preadipocytes.

Inadequate proliferation and/or differentiation of preadipocytes may lead to adipose tissue dysfunction characterized by hypertrophied, insulin-resistant adipocytes. Platelet-derived growth factor (PDGF) may alter adipose tissue function by promoting proliferation of preadipocytes. Two principal signaling pathways that regulate proliferation are PI3K/PI(3,4,5)P3/Akt and Shc/Ras/ERK1/2. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) dephosphorylates PI(3,4,5)P3, and also binds to Shc. Our goal was to determine how SHIP2 affects these PDGF signaling routes. To assess the role of the 5-phosphatase domain, we expressed wild-type or catalytically inactive dominant-negative SHIP2 (P686A-D690A-R691A; PDR/AAA) in 3T3-L1 preadipocytes. Surprisingly, SHIP2 PDR/AAA inhibited proliferation more potently than wild-type SHIP2. After three days of proliferation, phospho-Akt, phospho-ERK1/2, and PDGF receptor (PDGFR) levels were reduced in PDR/AAA-expressing preadipocytes. SHIP2 PDR/AAA interference with PDGFR signaling was demonstrated using imatinib, an inhibitor of PDGFR tyrosine kinase. The anti-proliferative effect of imatinib observed in control preadipocytes was not significant in SHIP2 PDR/AAA-expressing preadipocytes, indicating a pre-existing impairment of PDGFR-dependent mitogenesis in these cells. The inhibition of PDGF-activated mitogenic pathways by SHIP2 PDR/AAA was consistent with a decrease in PDGFR phosphorylation caused by a drop in receptor levels in SHIP2 PDR/AAA-expressing cells. SHIP2 PDR/AAA promoted ubiquitination of the PDGFR and its degradation via the lysosomal pathway independently of the association between the E3 ubiquitin ligase c-Cbl and PDGFR. Overall, our findings indicate that SHIP2 PDR/AAA reduces preadipocyte proliferation by attenuating PDGFR signaling.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation.

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

The directional response of chemotactic cells depends on a balance between cytoskeletal architecture and the external gradient.

Polarized migrating cells display signal transduction events, such as activation of phosphatidylinositol 3-kinase (PI3K) and Scar/Wave, and respond more readily to chemotactic stimuli at the leading edge. We sought to determine the basis of this polarized sensitivity. Inhibiting actin polymerization leads to uniform sensitivity. However, when human neutrophils were "stalled" by simultaneously blocking actin and myosin dynamics, they maintained the gradient of responsiveness to chemoattractant and also displayed noise-driven PIP3 flashes on the basal membrane, localized toward the front. Thus, polarized sensitivity does not require migration or cytoskeletal dynamics. The threshold for response is correlated with the static F-actin distribution, but not cell shape or volume changes, membrane fluidity, or the preexisting distribution of PI3K. The kinetics of responses to temporal and spatial stimuli were consistent with the local excitation global inhibition model, but the overall direction of the response was biased by the internal axis of polarity.

Assessment of development and chemotaxis in Dictyostelium discoideum mutants.

Studies using the social amoeba Dictyostelium discoideum have greatly contributed to the current understanding of the signaling network that underlies chemotaxis. Since directed migration is essential for normal D. discoideum multicellular development, mutants with chemotactic impairments are likely to have abnormal developmental morphologies. We have used multicellular development as a readout in a screen of mutants to identify new potential regulators of chemotaxis. In this chapter, we describe how mutants generated by restriction enzyme-mediated integration (REMI) are analyzed, from assessment of development to detailed characterization of 3',5'-cyclic adenosine monophosphate (cAMP)-induced responses. Two complementary approaches, plating cells either clonally on a bacterial lawn or as a population on non-nutrient agar, are used to evaluate multicellular development. Once mutants with aberrant developmental phenotypes are identified, their chemotaxis toward cAMP is assessed by both small population and micropipette assays. Furthermore, mutants are tested for defects in both general and specific signaling pathways by examining the recruitment of actin-binding LimE(Δcoil) or PIP3-binding PH domains to the plasma membrane in response to cAMP stimulation.

Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes.

The role of the inositol lipid 5-phosphatase (SHIP2) in preadipocyte signaling is not known. Although overexpression of SHIP2 inhibited proliferation and (3)H-thymidine incorporation in 3T3-L1 preadipocytes, there was no effect on insulin-induced adipogenesis. Insulin promoted SHIP2 tyrosine phosphorylation in differentiated 3T3-L1 adipocytes, but did not do so in preadipocytes. The absence of SHIP2 tyrosine phosphorylation suggests a potential explanation for the isolated rise in PI(3,4,5)P3, without any changes in PI(3,4)P2, previously observed following insulin treatment of these cells. Lack of SHIP2 tyrosine phosphorylation by insulin was also observed in primary cultures of human abdominal subcutaneous preadipocytes. These cells also produced PI(3,4,5)P3, but not PI(3,4)P2, in response to insulin. Comparison of insulin vs. PDGF treatment on SHIP2 tyrosine phosphorylation in 3T3-L1 and human preadipocytes revealed that only PDGF, which stimulates the accumulation of PI(3,4,5)P3 as well as PI(3,4)P2, was active in this regard, and only PDGF promoted the association of 52 kDa form of Shc with SHIP2. Nevertheless, both insulin and PDGF were equally effective in translocating SHIP2 to the plasma membrane in 3T3-L1 preadipocytes. Lack of SHIP2 tyrosine phosphorylation may account for the insulin-specific inositol phospholipid pattern of accumulation in preadipocytes.