Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface

UV Dose Governs UV-Polymerized Polyacrylamide Hydrogel Modulus

Polyacrylamide (PAA) hydrogels have become a widely used tool whose easily tunable mechanical properties, biocompatibility, thermostability, and chemical inertness make them invaluable in many biological applications, such as cell mechanosensitivity studies. Currently, preparation of PAA gels involves mixtures of acrylamide, bisacrylamide, a source of free radicals, and a chemical stabilizer. This method, while generally well accepted, has its drawbacks: long polymerization times, unstable and toxic reagents, and tedious preparation. Alternatively, PAA gels could be made by free radical polymerization (FRP) using ultraviolet (UV) photopolymerization, a method which is quicker, less tedious, and less toxic. Here, we describe a simple strategy based on total UV energy for determining the optimal UV crosslinking conditions that lead to optimal hydrogel modulus

Co-localization of myc-IQGAP1 mutants with WAVE2 and GFP-IQGAP1.

<p>B16F10 cells were co-transfected with a myc-tagged IQGAP1 mutant and GFP-IQGAP1-FL, and stained with anti-WAVE2. <b>A)</b> Representative images for myc-tagged IQGAP1 full length (FL) and mutant myc-tagged sequences (S1441E S1443D, S1441A S1443A, delta-CHD, delta-GRD and delta-CT) with corresponding GFP-IQGAP1-FL and WAVE2 images. <b>B)</b> Color-combine images of myc-tagged mutants (red), GFP-IQGAP1-FL (green) and WAVE2 (blue) with enlarged inset views of GFP-IQGAP1-FL positive and WAVE2 positive areas for each cell.</p

Distribution of microtubules in lamellipodia in cells expressing IQGAP1 mutants.

<p>A) Representative color combine images of IQGAP1 full length (FL) or IQGAP1 mutants (S1441E S1441D, S1441A S1443A, delta-CHD, delta-GRD and delta-CT) in green and microtubules in blue. B) Average number of microtubule ends within 2.1μm of the lamellipodium edge. The average edge length of lamellipodia was 80μm. ***p<0.001 Tukey’s post hoc test compared to FL. n = 11–15 cells for each bar. Bars = mean +/- s.e.m.</p

Polyacrylamide hydrogels composition and Young’s modulus.

<p>Polyacrylamide hydrogels composition and Young’s modulus.</p

Growth of B16F10 (F10), virus control (F10 VC), IQGAP1 knockdown (F10 KD) and F10 KD cells expressing GFP-IQGAP1-FL (F10 rKD) on laminin-coated PA hydrogels.

<p>Cells were added at low density and incubated for 4 d on 2D polyacrylamide hydrogels of 1, 10, or 100 kPa Young’s modulus coated with laminin. After incubation, samples were fixed and stained for DNA and actin. (A) Representative images for each condition show combined DNA (blue) and actin (green) channels. (B) Spheroid and (C) extra-spheroid growth of cells on laminin-coated PA hydrogels. The number of cell nuclei were counted inside the spheroid and in the area outside the spheroid within a 109 μm radius from the spheroid center (extra-spheroid). *p<0.05, **p<0.01, ***p<0.001 Tukey’s post hoc test. Bars = mean +/- s.e.m. n = 5–30 spheroids for each condition.</p

Effect of IQGAP1 knock down on cell morphology and cell division.

<p>Native B16F10 mouse melanoma cells (F10), virus control cells (F10 VC), IQGAP1 knock down cells (F10 KD) and F10 KD cells transfected with GFP-IQGAP1-FL (F10 rKD) were incubated on laminin-coated glass coverslips for 45 min (A-D) or in 12 well TCP plates for 4 d (E). (A) Representative images show individual nucleus, actin, IQGAP1 channels with combined nucleus (blue) and actin (green) images. The arrowhead marks a cell expressing GFP-IQGAP1. B) IQGAP1 protein quantified through immunofluorescence intensity. n = 38–40 cells for each condition. C) Projected cell area from actin images. n = 38–44 cells for each condition. D) Cell length expressed as the longest chord through the cell measured from actin images. n = 38–44 cells for each condition. E) Number of cells per cm² at 0, 1, 2, 3 and 4 d. n = 9 wells over 3 experiments for each time point. ***p<0.001 Tukey’s post hoc test as indicated by the brackets in C and D, or comparing F10 KD to F10 and F10 VC in B and E. Bars = mean +/- s.e.m. Time points = mean +/- 1 s.d.</p