Ali Kinkhabwala

Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling

Signal transduction through mitogen-activated protein kinase (MAPK) cascades is thought to occur through the assembly of macromolecular complexes. We quantified the abundance of complexes in the cytoplasm among the MAPKs Ste11, Ste7, Fus3 and the scaffold protein Ste5 in yeast pheromone signalling using fluorescence cross-correlation spectroscopy (FCCS). Significant complex concentrations were observed that remained unchanged on pheromone stimulation, demonstrating that global changes in complex abundances do not contribute to the transmission of signal through the cytoplasm. On the other hand, investigation of the distribution of active Fus3 (Fus3PP) across the cytoplasm using fluorescence lifetime imaging microscopy (FLIM) revealed a gradient of Fus3PP activity emanating from the tip of the mating projection. Spatial partitioning of Fus3 activating kinases to this site and deactivating phosphatases in the cytoplasm maintain this Fus3PP-activity distribution. Propagation of signalling from the shmoo is, therefore, spatially constrained by a gradient-generating reaction-diffusion mechanism.

Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.

Regulation of Ras Localization by Acylation Enables a Mode of Intracellular Signal Propagation

Active Ras is relayed between subcellular compartments by the acylation cycle. Ras Radio Waves The Ras family of small guanosine triphosphatases (GTPases) has roles in cellular proliferation and is frequently mutated in tumors. Stimulation of cells with epidermal growth factor leads to the transient activation of H-Ras at the plasma membrane, followed by an echo of this activity at the Golgi. This distinct spatiotemporal activity profile suggests that the Golgi is a passive receiver of Ras signals from the plasma membrane. Lorentzen et al. performed quantitative imaging and mathematical modeling of cells in which binding of H-Ras to GDP or GTP was decoupled from the acylation cycle that maintains its spatial organization. Regulation of H-Ras binding to GDP or GTP occurred only at the plasma membrane and not at the Golgi. Furthermore, the acylation cycle delivered active H-Ras from the plasma membrane to the Golgi, as well as from the endoplasmic reticulum to the Golgi; this latter delivery route enabled sustained H-Ras activity at the Golgi after the initial activation echo. Thus, the amount of active Ras at the Golgi is determined by activation of Ras at the plasma membrane and the endoplasmic reticulum, and the acylation cycle serves to relay activated Ras between subcellular compartments. Growth factor stimulation generates transient H-Ras activity at the plasma membrane but sustained activity at the Golgi. Two overlapping regulatory networks control compartmentalized H-Ras activity: the guanosine diphosphate–guanosine triphosphate cycle and the acylation cycle, which constitutively traffics Ras isoforms that can be palmitoylated between intracellular membrane compartments. Quantitative imaging of H-Ras activity after decoupling of these networks revealed regulation of H-Ras activity at the plasma membrane but not at the Golgi. Nevertheless, upon stimulation with epidermal growth factor, Ras activity at the Golgi displayed a pulse-like profile similar to that at the plasma membrane but also remained high after the initial stimulus. A compartmental model that included the acylation cycle and H-Ras regulation at the plasma membrane accounted for the pulse-like profile of H-Ras activity at the Golgi but implied that sustained H-Ras activity at the Golgi required H-Ras activation at an additional compartment, which we experimentally determined to be the endoplasmic reticulum. Thus, in addition to maintaining the localization of Ras, the acylation cycle underlies a previously unknown form of signal propagation similar to radio transmission in its generation of a constitutive Ras “carrier wave” that transmits Ras activity between subcellular compartments.

Dynamic recruitment of licensing factor Cdt1 to sites of DNA damage

For genomic integrity to be maintained, the cell cycle and DNA damage responses must be linked. Cdt1, a G1-specific cell-cycle factor, is targeted for proteolysis by the Cul4-Ddb1Cdt2 ubiquitin ligase following DNA damage. Using a laser nanosurgery microscope to generate spatially restricted DNA damage within the living cell nucleus, we show that Cdt1 is recruited onto damaged sites in G1 phase cells, within seconds of DNA damage induction. PCNA, Cdt2, Cul4, DDB1 and p21Cip1 also accumulate rapidly to damaged sites. Cdt1 recruitment is PCNA-dependent, whereas PCNA and Cdt2 recruitment are independent of Cdt1. Fitting of fluorescence recovery after photobleaching profiles to an analytic reaction-diffusion model shows that Cdt1 and p21Cip1 exhibit highly dynamic binding at the site of damage, whereas PCNA appears immobile. Cdt2 exhibits both a rapidly exchanging and an apparently immobile subpopulation. Our data suggest that PCNA provides an immobile binding interface for dynamic Cdt1 interactions at the site of damage, which leads to rapid Cdt1 recruitment to damaged DNA, preceding Cdt1 degradation.

Fluorescence fluctuations of quantum-dot sensors capture intracellular protein interaction dynamics

We extend the in vitro principle of co-immunoprecipitation to quantify dynamic protein interactions in living cells. Using a multiresolution implementation of fluorescence correlation spectroscopy to achieve maximal temporal resolution, we monitored the interactions of endogenous bait proteins, recruited by quantum dots, with fluorescently tagged prey. With this approach, we analyzed the rapid physiological regulation of protein kinase A.

Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.

Analytical model for macromolecular partitioning during yeast cell division.

BackgroundAsymmetric cell division, whereby a parent cell generates two sibling cells with unequal content and thereby distinct fates, is central to cell differentiation, organism development and ageing. Unequal partitioning of the macromolecular content of the parent cell which includes proteins, DNA, RNA, large proteinaceous assemblies and organelles can be achieved by both passive (e.g. diffusion, localized retention sites) and active (e.g. motor-driven transport) processes operating in the presence of external polarity cues, internal asymmetries, spontaneous symmetry breaking, or stochastic effects. However, the quantitative contribution of different processes to the partitioning of macromolecular content is difficult to evaluate.ResultsHere we developed an analytical model that allows rapid quantitative assessment of partitioning as a function of various parameters in the budding yeast Saccharomyces cerevisiae. This model exposes quantitative degeneracies among the physical parameters that govern macromolecular partitioning, and reveals regions of the solution space where diffusion is sufficient to drive asymmetric partitioning and regions where asymmetric partitioning can only be achieved through additional processes such as motor-driven transport. Application of the model to different macromolecular assemblies suggests that partitioning of protein aggregates and episomes, but not prions, is diffusion-limited in yeast, consistent with previous reports.ConclusionsIn contrast to computationally intensive stochastic simulations of particular scenarios, our analytical model provides an efficient and comprehensive overview of partitioning as a function of global and macromolecule-specific parameters. Identification of quantitative degeneracies among these parameters highlights the importance of their careful measurement for a given macromolecular species in order to understand the dominant processes responsible for its observed partitioning.

Implications of Network Topology on Stability

In analogy to chemical reaction networks, I demonstrate the utility of expressing the governing equations of an arbitrary dynamical system (interaction network) as sums of real functions (generalized reactions) multiplied by real scalars (generalized stoichiometries) for analysis of its stability. The reaction stoichiometries and first derivatives define the network’s “influence topology”, a signed directed bipartite graph. Parameter reduction of the influence topology permits simplified expression of the principal minors (sums of products of non-overlapping bipartite cycles) and Hurwitz determinants (sums of products of the principal minors or the bipartite cycles directly) for assessing the network’s steady state stability. Visualization of the Hurwitz determinants over the reduced parameters defines the network’s stability phase space, delimiting the range of its dynamics (specifically, the possible numbers of unstable roots at each steady state solution). Any further explicit algebraic specification of the network will project onto this stability phase space. Stability analysis via this hierarchical approach is demonstrated on classical networks from multiple fields.

Journey to the Center of the Mitochondria Guided by the Tail Anchor of Protein Tyrosine Phosphatase 1B

The canonical protein tyrosine phosphatase PTP1B has traditionally been considered to exclusively reside on the endoplasmic reticulum (ER). Using confocal microscopy, we show that endogenous PTP1B actually exhibits a higher local concentration at the mitochondria in all mammalian cell lines that we tested. Fluorescently labeled chimeras containing full-length PTP1B or only its 35 amino acid tail anchor localized identically, demonstrating the complete dependence of PTP1B’s subcellular partitioning on its tail anchor. Correlative light and electron microscopy using GFP-driven photo-oxidation of DAB revealed that PTP1B’s tail anchor localizes it to the mitochondrial interior and to mitochondrial-associated membrane (MAM) sites along the ER. Heterologous expression of the tail anchor of PTP1B in the yeast S. cerevisiae surprisingly led to its exclusive localization to the ER/vacuole with no presence at the mitochondria. Studies with various yeast mutants of conserved membrane insertion pathways revealed a role for the GET/TRC40 pathway in ER insertion, but also emphasized the likely dominant role of spontaneous insertion. Further studies of modified tail isoforms in both yeast and mammalian cells revealed a remarkable sensitivity of subcellular partitioning to slight changes in transmembrane domain (TMD) length, C-terminal charge, and hydropathy. For example, addition of a single positive charge to the tail anchor was sufficient to completely shift the tail anchor to the mitochondria in mammalian cells and to largely shift it there in yeast cells, and a point mutation that increased TMD hydropathy was sufficient to localize the tail anchor exclusively to the ER in mammalian cells. Striking differences in the subcellular partitioning of a given tail anchor isoform in mammalian versus yeast cells most likely point to fundamental differences in the lipid composition of specific organelles (e.g. affecting membrane charge or thickness) in higher versus lower eukaryotes. Fluorescence lifetime imaging microscopy (FLIM) detection of the Förster Resonance Energy Transfer (FRET)-based interaction of the catalytic domain of PTP1B with the epidermal growth factor receptor (EGFR/ErbB1) at the mitochondria revealed a strong interaction on the cytosolic face of the outer mitochondrial membrane (OMM), suggesting the presence of a significant pool of PTP1B there and a novel role for PTP1B in the regulation of mitochondrial ErbB1 activity. In summary, in addition to its well-established general localization along the ER, our results reveal that PTP1B specifically accumulates at MAM sites along the ER and localizes as well to the OMM and mitochondrial matrix. Further elucidation of PTP1B’s roles in these different locations (including the identification of its targets) will likely be critical for understanding its complex regulation of general cellular responses, cell proliferation, and diseased states.