Ulcerative Skin Lesions

Abstract A 69 year old Caucasian female smoker with an unremarkable past medical history presenting with diffusing necrotic and ulcerative skin lesions of unknown etiology. After an extensive workup, the diagnosis of calciphylaxis sine calcifications was made following results from laboratory and biopsy results

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

<div><p>F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. <i>Dictyostelium</i> myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure <i>in situ</i>. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.</p></div

Time-course of the appearance of actin waves in cells starved for short times.

<p>The percentages of cells with waves are shown at the starvation times (min) indicated in the figure. Short movies (10 frames every 10 s) of randomly chosen fields were recorded. Cells in each field were scored for the presence of waves visualized with mRFP-lifeact. (A) Comparison of <i>myoB⁻</i>-cells (filled circles) and the parent WT-AX2 cells (open circles). Both cell lines were expressing mRFP-lifeact. (B) Comparison of <i>myoB⁻</i>-cells expressing either mRFP-lifeact alone (filled circles) or mRFP-lifeact and GFP-MIB (open circles). The two cell lines compared in A and B were grown and treated identically and recorded in the same experiment so the two cell lines within each panel can be directly compared with each other. The results are representative of two independent experiments. Error bars were calculated using the binomial probability confidence interval calculator developed by Daniel S. Soper (<a href="http://www.danielsoper.com/statcalc" target="_blank">http://www.danielsoper.com/statcalc</a>) and correspond to the 95% confidence interval.</p

MIB mutants used in the current study.

<p>MIB mutants used in the current study.</p

MIB localizes at the front of a moving actin wave in <i>myoB⁻</i>-cells expressing GFP-MIB and mRFP-lifeact.

<p>The left panel shows the line scans of the fluorescence intensity of GFP-MIB and mRFP-lifeact of the cell shown in the right panel. The line scans correspond to the boxed area within the cell and the red arrow in the box indicates the direction of scanning. 0 nm in the line scan corresponds to the beginning of the box. The direction of wave movement is indicated in the left panel. Cells were starved for 30 min after which 1 µM latrunculin was added and cell images were recorded. Bar is 10 µm.</p

The GPQ-region is required for localization of MIB to protrusions on the dorsal cell membrane.

<p><i>myoB⁻</i>-cells co-transfected with mRFP-lifeact and either GFP-MIB or GFP-tagged MIB mutants, as indicated in the figure, were starved for 2 h and monitored for the presence of MIB and its mutants in the actin-rich protrusions on the dorsal cell membrane. These images were taken at a higher focal plane than images visualizing actin waves on the ventral surface. Arrows point to actin-rich protrusions. Bar is 10 µm.</p

Actin binding through the head contributes to MIB association with waves.

<p><i>myoB⁻</i>-cells were co-transfected with mRFP-lifeact and either GFP-MIB or GFP-tagged N154A, N154A/BH-Ala, E407K or S322A, as indicated in the figure. Cells were starved for 30 min after which 1 µM latrunculin was added and cell images were recorded. Bar is 10 µm.</p

Expression of lifeact does not affect localization of MIB in <i>myoB⁻</i>-cells.

<p><i>myoB⁻</i>-cells were co-transfected with mRFP-lifeact and GFP-MIB, and the localization of F-actin and MIB were monitored in live cells. MIB co-localized with F-actin: (A) at the plasma membrane of freshly plated cells; (B) in protrusions of cells starved for 1–2 h; (C) at cell-cell contacts of cells starved for 1–2 h; and (D) at the front of elongated cells moving directionally. The arrows point to the sites enriched in MIB. Bar is 10 µm.</p

The GPQ-region is essential for MIB association with actin waves.

<p><i>myoB⁻</i>-cells were co-transfected with mRFP-lifeact and with either GFP-MIB or GFP-tagged dGPQ/SH3, dGPQ, dSH3 or Tail as indicated in the figure. Cells were starved for 30 min after which 1 µM latrunculin was added and cell images were recorded. Mutants missing the GPQ region (dGPQ and dGPQ/SH3) did not associate with waves whereas dSH3 and Tail did. Bar is 10 µm.</p