Michael A. Ferenczi

Cdc42 and Par6–PKCζ regulate the spatially localized association of Dlg1 and APC to control cell polarization

Cell polarization is essential in a wide range of biological processes such as morphogenesis, asymmetric division, and directed migration. In this study, we show that two tumor suppressor proteins, adenomatous polyposis coli (APC) and Dlg1-SAP97, are required for the polarization of migrating astrocytes. Activation of the Par6–PKCζ complex by Cdc42 at the leading edge of migrating cells promotes both the localized association of APC with microtubule plus ends and the assembly of Dlg-containing puncta in the plasma membrane. Biochemical analysis and total internal reflection fluorescence microscopy reveal that the subsequent physical interaction between APC and Dlg1 is required for polarization of the microtubule cytoskeleton.

ATPase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres: a real time phosphate assay

The rate of appearance of inorganic phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3‐l‐(2‐nitrophenyl)ethyl ester of ATP (NPE‐caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pisensitive probe, MDCC‐PBP, a coumarin‐labelled A197C mutant of the phosphate‐binding protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH‐linked coupled enzyme assay. On photolysis of NPE‐caged ATP in the presence of Ca2+ at 20°C, the fluorescence increase of MDCC‐PBP was non‐linear with time. ATPase activity was 41 s−1 in the first turnover based on a myosin subfragment 1 concentration of 150 μm. This was calculated from a linear regression of the fluorescence signal reporting 20‐150 μm of Pi release. Tension was at 67 % of its isometric level by the time 150 μm Pi was released. ATPase activities were 36 and 31 s−1for Pi released in the ranges of 150‐300μM and 300‐450 μm, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20°C. An NADH‐linked assay showed the ATPase activity had a lower limit of 12.7 s−1 at 20°C. The response to photoly tic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s−1. On photolysis of NPE‐caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s−1 at 20°C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl‐EGTA. At 5°C the ATPase rate was 5.8 and 4.0 s−1 in the first and second turnovers, respectively. These rates are comparable to those when NPE‐caged ATP was used. The influence of ADP and Pi on the ATPase activities was measured using the MDCC‐PBP and NADH‐linked assays, respectively. ADP (0.5 him) decreased the initial ATPase rate by 23%. Pi (10 him) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. The MDCC‐PBP assay and NPE‐caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5°C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s−1 during the first turnover (based on 180 μm myosin subfragment 1). Tension was 74% of its isometric level by the time 180 μm Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.

Interaction of the actin cytoskeleton with microtubules regulates secretory organelle movement near the plasma membrane in human endothelial cells

The role of cytoskeletal elements in regulating transport and docking steps that precede exocytosis of secretory organelles is not well understood. We have used Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the three-dimensional motions of secretory organelles near the plasma membrane in living endothelial cells. Weibel-Palade bodies (WPb), the large tubular storage organelles for von Willebrand factor, were labelled with Rab27a-GFP. By contrast, green fluorescent protein (GFP)-tagged tissue-type plasminogen activator (tPA-GFP) labelled submicron vesicular organelles. Both populations of GFP-labelled organelles underwent stimulated exocytosis. The movement of these morphologically distinct organelles was measured within the evanescent field that penetrated the first 200 nm above the plasma membrane. WPb and tPA-GFP vesicles displayed long-range bidirectional motions and short-range diffusive-like motions. Rotating and oscillating WPb were also observed. TIRF microscopy enabled us to quantify the contribution of actin and microtubules and their associated motors to the organelle motions close to the plasma membrane. Long-range motions, as well as WPb rotations and oscillations, were microtubule-and kinesin-dependent. Disruption of the actin cytoskeleton and inhibition of myosin motors increased the number of long-range motions and, in the case of WPb, their velocity. The actin and microtubules had opposite effects on the mobility of organelles undergoing short-range motions. Actin reduced the mobility and range of motion of both WPb and tPA vesicles, whereas microtubules and kinesin motors increased the mobility of WPb. The results show that the dynamics of endothelial secretory organelles close to the plasma membrane are controlled by the opposing roles of the microtubule and actin cytoskeletal transport systems.

Phosphate burst in permeable muscle fibers of the rabbit

The transient kinetics of ATP hydrolysis in chemically skinned psoas muscle fibers of the rabbit have been measured. Muscles fibers in the rigor state (absence of nucleotide) were relaxed rapidly by the photochemical release of [2-3H]ATP from caged-ATP (P3-1-(2-nitro)phenylethyl[2-3H]adenosine 5-triphosphate) in the absence of calcium ions. Rapid freezing of the fiber to stop hydrolysis, followed by analysis of the tritiated nucleotide content allowed the course of the hydrolysis to be determined. The timecourse of ATP hydrolysis was biphasic, with an initial rapid phase occurring at a rate of approximately 60 s-1 at 12 degrees C for fibers exposed to greater than 0.7 mM ATP. The amplitude of the rapid phase was as previously reported (Ferenczi, M. A., E. Homsher, and D. R. Trentham, 1984, J. Physiol. (Lond.)., 352:575-599).

Rab27b Regulates Mast Cell Granule Dynamics and Secretion

The Rab GTPase family regulates membrane domain organization and vesicular transport pathways. Recent studies indicate that one member of the family, Rab27a, regulates transport of lysosome‐related organelles in specialized cells, such as melanosomes and lytic granules. Very little is known about the related isoform, Rab27b. Here we used genetically modified mice to study the involvement of the Rab27 proteins in mast cells, which play key roles in allergic responses. Both Rab27a and Rab27b isoforms are expressed in bone marrow‐derived mast cells (BMMC) and localize to secretory granules. Nevertheless, secretory defects as measured by β‐hexosaminidase release in vitroand passive cutaneous anaphylaxis in vivowere found only in Rab27b and double Rab27 knockout (KO) mice. Immunofluorescence studies suggest that a subset of Rab27b and double Rab27‐deficient BMMCs exhibit mild clustering of granules. Quantitative analysis of live‐cell time‐lapse imaging revealed that BMMCs derived from double Rab27 KO mice showed almost 10‐fold increase in granules exhibiting fast movement (>1.5 μm/s), which could be disrupted by nocodazole. These results suggest that Rab27 proteins, particularly Rab27b, play a crucial role in mast cell degranulation and that their action regulates the transition from microtubule to actin‐based motility.

Structural Changes in the Actin–Myosin Cross-Bridges Associated with Force Generation Induced by Temperature Jump in Permeabilized Frog Muscle Fibers

Structural changes induced by Joule temperature jumps (T-jumps) in frog muscle fibers were monitored using time-resolved x-ray diffraction. Experiments made use of single, permeabilized fibers that were fully activated after slight cross-linking with 1-ethyl-3-[3-dimethylamino)propyl]carbodiimide to preserve their structural order. After T-jumps from 5-6 to approximately 17 degrees C and then on to approximately 30 degrees C, tension increased by a factor of 1.51 and 1.84, respectively, whereas fiber stiffness did not change with temperature. The tension rise was accompanied by a decrease in the intensity of the (1, 0) equatorial x-ray reflection by 15 and 26% (at approximately 17 and approximately 30 degrees C) and by an increase in the intensity of the M3 myosin reflection by 20% and 41%, respectively. The intensity of the (1,1) equatorial reflection increased slightly. The peak of the intensity on the 6th actin layer line shifted toward the meridian with temperature. The intensity of the 1st actin layer line increased from 12% (of its rigor value) at 5-6 degrees C to 36% at approximately 30 degrees C, so that the fraction of the cross-bridges labeling the actin helix estimated from this intensity increased proportionally to tension from approximately 35% at 5-6 degrees C to approximately 60% at approximately 30 degrees C. This suggests that force is generated during a transition of nonstereo-specifically attached myosin cross-bridges to a stereo-specific binding state.

Physiological properties of human diaphragm muscle fibres and the effect of chronic obstructive pulmonary disease

The contractile and actomyosin ATPase properties of single fibres were examined in human diaphragm muscle obtained from patients with and without chronic obstructive pulmonary disease (COPD). Costal diaphragm biopsies were taken from five patients without evidence of COPD and from 11 age‐matched individuals with varying degrees of the disease. Our aim was to establish whether changes in contractile properties of COPD diaphragm could be fully explained by the previously documented shift towards a greater proportion of type I myosin heavy chain isoform in COPD. The relative proportion of type I diaphragm fibres from non‐COPD and COPD patients was measured by gel electrophoresis, and was negatively correlated with FEV1 over the full range of values investigated. There was also significant atrophy of the type I fibre population in COPD diaphragms. Isometric tension was similar among the fibre types and between the COPD and non‐COPD patients. The intrinsic energetic properties of diaphragm fibres were examined by monitoring the time‐resolved actomyosin ATPase activity in COPD and non‐COPD fibres that produced similar isometric forces. The isometric ATPase rate in COPD fibres was reduced to 50% of the rate in non‐COPD fibres; hence, the cost of isometric contraction in type I and type IIA COPD fibres was reduced to between one‐third and one‐half of the tension cost calculated for non‐COPD fibres. The rate of force development in type I COPD fibres was reduced to 50% of the rate seen in non‐COPD type‐I fibres. No difference in the rate of ATP consumption between COPD and non‐COPD fibres was evident during isovelocity shortening. These data extend previous findings showing that aspects of breathing mechanics during progressive COPD are associated with remodelling of the diaphragm fibre‐type distribution; on top of the increase in type I fibres there are fibre‐specific reductions in force development rate (type I fibres) and ATPase rate that are consistent with the impairment of cross‐bridge cycling kinetics.

Rab27a and MyoVa are the primary Mlph interactors regulating melanosome transport in melanocytes

Melanosome transport in melanocytes is a model system for the study of cytoskeletal regulation of intracellular transport. Melanophilin (Mlph) is a Rab27a- and myosin Va (MyoVa)-binding protein that regulates this process. Using yeast two-hybrid screening, we identified MT plus-end binding protein (EB1) as a melanocyte-expressed Mlph-interacting protein. To address the role of EB1 versus Rab27a and MyoVa interactions in Mlph targeting and function, we used siRNA and Mlph mutations to specifically disrupt each interaction in cultured melanocytes. Using the Mlph R35W mutant that blocks Mlph-Rab27a interaction and Rab27a siRNA we show this interaction is required for melanosome targeting and stability of Mlph. Mutants and siRNA that affect Mlph-MyoVa and Mlph-EB1 interactions reveal that while neither MyoVa nor EB1 affect Mlph targeting to melanosomes, MyoVa but not EB1 interaction is required for transport of melanosomes to peripheral dendrites. We propose that Mlph is targeted to and/or stabilised on melanosomes by Rab27a, and then recruits MyoVa, which provides additional stability to the complex and allows melanosomes to transfer from MT to actin-based transport and achieve peripheral distribution. EB1 appears to be non-essential to this process in cultured melanocytes, which suggests that it plays a redundant role and/or is required for melanocyte/keratinocyte contacts and melanosome transfer.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Background: Cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle function. Results: Phosphorylation affects mechanical parameters of cardiac muscle contraction during shortening. Conclusion: Phosphorylation impacts mechanical function of cardiac muscle and is altered during cardiac disease. Significance: Understanding RLC regulation by phosphorylation in cardiac muscle contraction is crucial for understanding changes in disease. Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes.

Acetylcholinesterase (AChE) terminates neurotransmission at cholinergic synapses by hydrolysing acetylcholine, but also has non‐enzymatic morphoregulatory effects on neurons such as stimulation of neurite outgrowth. It is widely expressed outside the nervous system, but its function in non‐neuronal cells is unclear. Here we have investigated the distribution and function of AChE in fibroblasts and astrocytes. We show that these cells express high levels of AChE protein that co‐migrates with recombinant AChE but contains little catalytic activity. Fibroblasts express transcripts encoding the synaptic AChE‐T isoform and its membrane anchoring peptide PRiMA‐I. AChE is strikingly distributed in arcs, rings and patches at the leading edge of spreading and migrating fibroblasts and astrocytes, close to the cell–substratum interface, and in neuronal growth cones. During in vivo healing of mouse skin, AChE becomes highly expressed in re‐epithelialising epidermal keratinocytes 1 day after wounding. AChE appears to be functionally important for polarised cell migration, since an AChE antibody reduces substratum adhesion of fibroblasts, and slows wound healing in vitro as effectively as a β1‐integrin antibody. Moreover, elevation of AChE expression increases fibroblast wound healing independently of catalytic activity. Interestingly, AChE surface patches precisely co‐localise with amyloid precursor protein and the extracellular matrix protein perlecan, but not focal adhesions or α‐dystroglycan, and contain a high concentration of tyrosine phosphorylated proteins in spreading cells. These findings suggest that cell surface AChE, possibly in a novel signalling complex containing APP and perlecan, contributes to a generalised mechanism for polarised membrane protrusion and migration in all adherent cells. J. Cell. Physiol. 215: 82–100, 2008.