Colloidal Nanoribbons: From Infrared to Visible.

Using the cation-exchange method, colloidal PbS nanoribbons are converted completely into CdS nanoribbons. This process expands the emission spectrum of the nanoribbons from infrared to visible. The morphology of nanoribbons remains the same after cation exchange, but the crystal structure changes from rock salt to zincblende. CdS nanoribbons exhibit blue band-edge photoluminescence under ultraviolet-light excitation. Cathodoluminescence spectroscopy of the CdS nanoribbons shows multicolor (blue, green, and red) emissions. Further time-resolved photoluminescence spectroscopy studies show that the lifetime of the midgap states is more than 2 orders of magnitude longer than that of the band-edge states.

Activated Protein Kinase C (PKC) Is Persistently Trafficked with Epidermal Growth Factor (EGF) Receptor.

Protein kinase Cs (PKCs) are activated by lipids in the plasma membrane and bind to a scaffold assembled on the epidermal growth factor (EGF) receptor (EGFR). Understanding how this complex is routed is important, because this determines whether EGFR is degraded, terminating signaling. Here, cells were preincubated in EGF-tagged gold nanoparticles, then allowed to internalize them in the presence or absence of a phorbol ester PKC activator. PKC colocalized with EGF-tagged nanoparticles within 5 min and migrated with EGFR-bearing vesicles into the cell. Two conformations of PKC-epsilon were distinguished by different primary antibodies. One, thought to be enzymatically active, was on endosomes and displayed a binding site for antibody RR (R&D). The other, recognized by Genetex green (GG), was soluble, on actin-rich structures, and loosely bound to vesicles. During a 15-min chase, EGF-tagged nanoparticles entered large, perinuclear structures. In phorbol ester-treated cells, vesicles bearing EGF-tagged nanoparticles tended to enter this endocytic recycling compartment (ERC) without the GG form. The correlation coefficient between the GG (inactive) and RR conformations on vesicles was also lower. Thus, active PKC has a Charon-like function, ferrying vesicles to the ERC, and inactivation counteracts this function. The advantage conferred on cells by aggregating vesicles in the ERC is unclear.

The tumor promoter-activated protein kinase Cs are a system for regulating filopodia.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, ß, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter-stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/ß pseudosubstrate sequence, and an inhibitor of T-cell protein tyrosine phosphatase (TC-PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter-treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ-mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady-state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long-term "memory" of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

Effect of Cdc42 domains on filopodia sensing, cell orientation, and haptotaxis.

Filopodia are sensors which, along with microtubules, regulate the persistence of locomotion. To determine whether protrusions were involved in sensing adhesion, epithelial cells were cultured on platinum and tantalum gradients. Protrusions were defined by an unbiased statistical method of classification as factors 4 (filopodia), 5 (mass distribution), and 7 (nascent neurites). When the prevalence of protrusions was measured in zones of high (H), middle (M), and low (L) adhesiveness, the main differences were in factor 4. Its values were highest at H and declined at M and L regardless of the gradient composition. The significance of the differences was enhanced when T (top/adhesive end) and B (bottom/nonadhesive end) sides of cells were analyzed separately. Since information about sidedness increased the statistical power of the test, this result suggested that cells pointed more filopodia toward the adhesive end. Trends occurred in factors 5 and 7 only when conditions allowed for a marked trend in factor 4. The data showed that gradient sensing is proportional to the prevalence of filopodia, and filopodia are the only protrusions engaged in comparing adhesiveness across a cell. The probability (P) of the significance of a trend was then used to determine how cells sense the gradient. Binding peptides (BPs) were introduced representing sequences critical for Cdc42 docking on a specific partner. BPs for IQGAP (IQ(calmodulin-binding domain)-containing GTPase-activating protein) and ACK (Cdc42-associated kinase) reduced factor 4 values and prevented cell orientation on the gradient. Micrographs showed attenuated or stubby filopodia. These effectors may be implicated in gradient sensing. Another IQGAP BP increased filopodia prevalence and enhanced orientation on the gradient (P<0.00015). A Wiskott-Aldrich syndrome protein (WASP) BP had no effect. When sensing and orientation were abolished, they both failed at the level of filopodia, indicating that filopodia are both sensors and implementers of signals transduced by adhesion.

Unraveling the determinants of protrusion formation.

A COMPUTERIZED MORPHOMETRIC CLASSIFICATION TECHNIQUE BASED ON LATENT FACTORS REVEALS MAJOR PROTRUSION CLASSES: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.

Morphological fractal analysis of shape in cancer cells treated with combinations of microtubule-polymerizing and -depolymerizing agents.

The current prognostic parameters, including tumor volume, biochemistry, or immunohistochemistry, are not sufficient to reflect the properties of cancer cells that distinguish them from normal cells. Our focus is to evaluate the effects of a combination of microtubule-polymerizing Taxol and -depolymerizing colchicine on IAR20 PC1 liver cells by measuring the surface fractal dimension as a descriptor of two-dimensional vascular geometrical complexity. The fractal dimension offers a rapid means of assessing cell shape. Furthermore, we show correlations of fractal dimensions of cell contours with the latent factors from our previously employed cell shape analysis.

Relationship of p21-activated kinase (PAK) and filopodia to persistence and oncogenic transformation.

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

Contact inhibition revisited.

Contact inhibition of cell movement was originally defined in the 1950s as a way of interpreting studies that were ethological and statistical in nature. Research done in succeeding decades provided a more detailed study of the initial contact and its consequences for the cell. The behavior called contact inhibition is characterized by the cessation of ruffling and forward movement in the lamellipodium of the cell making the contact. A new ruffling membrane then arises elsewhere on the cell perimeter. A comparison between the contact behavior described in the early literature and that of the nerve growth cone, described recently by Steketee and Tosney, suggests that filopodia mediate the sensing function in both cases. Since transformed cells have fewer filopodia than normal cells, the contact behavior may decline in direct response to the degraded function of filopodia. This new "filopodia focal signal transduction" hypothesis of contact inhibition elevates the filopodia sensing function and the cessation of lamellipodial advance to the highest importance as phenomena underlying the altered behavior of cancer cells.

Actin-based features negatively regulated by protein kinase C-epsilon.

Cells exposed to phorbol 12-myristate 13-acetate (PMA) undergo a choreographed sequence of morphological changes. Some of these, including stimulation of membrane ruffles and the later appearance of stress fibers, rely on remodeling of the actin cytoskeleton. Although this process is poorly understood, it is important, because the same features are affected during oncogenic transformation. PMA also activates protein kinase C (PKC). Enzyme activation is followed by degradation. Either process might affect the remodeling of actin. The present studies determined whether any PKC isozymes were subject to degradation in tracheal epithelial cells by quantifying the amount of each isozyme present after PMA exposure. PKC-epsilon was the only isozyme to show declining content correlated with increased stress fiber accumulation. Stress fibers increased between 5 and 10 h, whereas PKC-epsilon declined to 38% of its starting value (95% confidence interval, 10-68%). The relationship could be fit by the function F(x) = 0.683 x exp[-0.841(x - 0.387)], where F is the frequency of fiber-containing cells and x is PKC-epsilon content. Fiber accumulation was further investigated after knockdown of PKC-epsilon with RNA interference and antisense oligodeoxynucleotide. Knockdown enhanced stress fibers in cells not yet exposed to PMA as well as the final frequency of fiber-containing cells after PMA exposure. With knockdown at both transcriptional and protein levels, approximately 15% of the original content was predicted and achieved, as judged from real-time PCR and PKC-epsilon content measurements. The results suggest that PKC-epsilon negatively regulates stress fibers, either by directly turning over one of their components or by regulating an upstream step affecting fiber organization.

Colchitaxel, a coupled compound made from microtubule inhibitors colchicine and paclitaxel.

BACKGROUND: Tumor promoters enhance tumor yield in experimental animals without directly affecting the DNA of the cell. Promoters may play a role in the development of cancer, as humans are exposed to them in the environment. In work based on computer-assisted microscopy and sophisticated classification methods, we showed that cells could be classified by reference to a database of known normal and cancerous cell phenotypes. Promoters caused loss of properties specific to normal cells and gain of properties of cancer cells. Other compounds, including colchicine, had a similar effect. Colchicine given together with paclitaxel, however, caused cells to adopt properties of normal cells. This provided a rationale for tests of microtubule inhibitor combinations in cancer patients. The combination of a depolymerizing and a stabilizing agent is a superior anti-tumor treatment. The biological basis of the effect is not understood. RESULTS: A single compound containing both colchicine and paclitaxel structures was synthesized. Colchicine is an alkaloid with a trimethoxyphenyl ring (ring A), a ring with an acetamide linkage (ring B), and a tropolone ring (ring C). Although rings A and C are important for tubulin-binding activity, the acetamide linkage on ring B could be replaced by an amide containing a glutamate linker. Alteration of the C-7 site on paclitaxel similarly had little or no inhibitory effect on its biological activity. The linker was attached to this position. The coupled compound, colchitaxel (1), had some of the same effects on microtubules as the combination of starting compounds. It also caused shortening and fragmentation of the + end protein cap. CONCLUSION: Since microtubule inhibitor combinations give results unlike those obtained with either inhibitor alone, it is important to determine how such combinations affect cell shape and growth. Colchitaxel shows a subset of the effects of the inhibitor combination. Thus, it may be able to bind the relevant cellular target of the combination. It will be useful to determine the basis of the shape reversal effect and possibly, the reasons for therapeutic efficacy of microtubule inhibitor combinations.