Jeffrey Drue David

Sequential activation of caspases and serine proteases (serpases) during apoptosis.

Analogous to caspases, serine (Ser) proteases are involved in protein degradation during apoptosis. It is unknown, however, whether Ser proteases are activated concurrently, sequentially, or as an alternative to the activation of caspases. Using fluorescent inhibitors of caspases (FLICA) and Ser proteases (FLISP), novel methods to detect activation of of these enzymes in apoptotic cells, we demonstrate that two types of Ser protease sites become accessible to these inhibitors during apoptosis of HL-60 cells. The prior exposure to caspases inhibitor Z-VAD-FMK markedly diminished activation of both Ser protease sites. However, the unlabeled inhibitor of Ser-proteases TPCK had modest suppressive effect- while TLCK had no effect- on the activation of caspases. Activation of caspases, thus, appears to be an upstream event and likely a prerequisite for activation of FLISP- reactive sites. Differential labeling with the red fluorescing sulforhodamine-tagged VAD-FMK and the green fluorescing FLISP allowed us to discriminate, within the same cell, between activation of caspases and Ser protease sites. Despite a certain degree of co-localization, the pattern of intracellular caspase- vs FLISP- reactive sites, was different. Also different were relative proportions of activated caspases vs Ser protease sites in individual cells. The observed induction of FLISP-binding sites we interpret as revealing activation of at least two different apoptotic Ser proteases; by analogy to caspases we denote them serpases. Their apparent molecular weight (62-65 kD) suggests that they are novel enzymes. Key Words: Serpases, Caspases, Apoptosis, Enzymatic center, Chymotrypsin, FLICA, FLISP, Cell necrobiology

Using Wafer-Scale Patterns for CMP Analysis

A new set of wafer-scale patterns has been designed for analysis and modeling of key CMP effects. In particular, the goal of this work is to develop methods to characterize the planarization capability of a CMP process using simple measurements on wafer scale patterns. We examine means to pattern large trenches (e.g. 1 to 15 mm wide and 15 mm tall) or circles across 4” and 8” wafers, and present oxide polish results using both stacked and solo pads in conventional polish processes. We find that large separation (15 mm) between trenches enables cleaner measurement and analysis. Examination of oxide removal in the center of the trench as a function of trench width shows a saturation at a length comparable to the planarization length extracted from earlier studies of small-scale oxide patterns. Increase in polish pressure is observed to decrease this saturation point. Such wafer scale patterns may provide information on pad flexing limits in addition to planarization length, and promise to be useful in both patterned wafer CMP modeling and studies of wafer scale CMP dependencies such as nanotopography.