Lamellipodia are crucial for haptotactic sensing and response.

Haptotaxis is the process by which cells respond to gradients of substrate-bound cues, such as extracellular matrix proteins (ECM); however, the cellular mechanism of this response remains poorly understood and has mainly been studied by comparing cell behavior on uniform ECMs with different concentrations of components. To study haptotaxis in response to gradients, we utilized microfluidic chambers to generate gradients of the ECM protein fibronectin, and imaged the cell migration response. Lamellipodia are fan-shaped protrusions that are common in migrating cells. Here, we define a new function for lamellipodia and the cellular mechanism required for haptotaxis - differential actin and lamellipodial protrusion dynamics lead to biased cell migration. Modest differences in lamellipodial dynamics occurring over time periods of seconds to minutes are summed over hours to produce differential whole cell movement towards higher concentrations of fibronectin. We identify a specific subset of lamellipodia regulators as being crucial for haptotaxis. Numerous studies have linked components of this pathway to cancer metastasis and, consistent with this, we find that expression of the oncogenic Rac1 P29S mutation abrogates haptotaxis. Finally, we show that haptotaxis also operates through this pathway in 3D environments.

Mesenchymal chemotaxis requires selective inactivation of myosin II at the leading edge via a noncanonical PLCγ/PKCα pathway.

Chemotaxis, migration toward soluble chemical cues, is critical for processes such as wound healing and immune surveillance and is exhibited by various cell types, from rapidly migrating leukocytes to slow-moving mesenchymal cells. To study mesenchymal chemotaxis, we observed cell migration in microfluidic chambers that generate stable gradients of platelet-derived growth factor (PDGF). Surprisingly, we found that pathways implicated in amoeboid chemotaxis, such as PI3K and mammalian target of rapamycin signaling, are dispensable for PDGF chemotaxis. Instead, we find that local inactivation of Myosin IIA, through a noncanonical Ser1/2 phosphorylation of the regulatory light chain, is essential. This site is phosphorylated by PKCα, which is activated by an intracellular gradient of diacylglycerol generated by PLCγ. Using a combination of live imaging and gradients of activators/inhibitors in the microfluidic chambers, we demonstrate that this signaling pathway and subsequent inhibition of Myosin II activity at the leading edge are required for mesenchymal chemotaxis.

Association of chronic obstructive pulmonary disease severity and Pneumocystis colonization.

Factors modulating the variable progression of chronic obstructive pulmonary disease (COPD) are largely unknown, but infectious agents may play a role. Because Pneumocystis has previously been shown to induce a CD8(+) lymphocyte- and neutrophil-predominant response similar to that in COPD, we explored the association of the organism with accelerated disease progression. We examined Pneumocystis colonization rates in lung tissue obtained during lung resection or transplantation in smokers with a range of airway obstruction severity and in a control group with lung diseases other than COPD. Using nested polymerase chain reaction, Pneumocystis colonization was detected in 36.7% of patients with very severe COPD (Global Health Initiative on Obstructive Lung Disease [GOLD] Stage IV) compared with 5.3% of smokers with normal lung function or less severe COPD (Stages 0, I, II, and III) (p = 0.004) and with 9.1% of control subjects (p = 0.007). Colonized subjects exhibited more severe airway obstruction (median FEV(1) = 21% predicted versus 62% in noncolonized subjects, p = 0.006). GOLD IV was the strongest predictor of Pneumocystis colonization (odds ratio = 7.3, 95% confidence interval = 2.4-22.4, p < 0.001) and was independent of smoking history. We conclude that there is a strong association between Pneumocystis colonization and severity of airflow obstruction in smokers, suggesting a possible pathogenic link with COPD progression.

Prevalence and clinical predictors of Pneumocystis colonization among HIV-infected men.

BACKGROUND: The epidemiology and transmission of Pneumocystis are poorly understood. The incidence of colonization, or detection of organisms without signs of disease, has been debated, and risk factors for colonization are largely unknown. OBJECTIVE: To determine the rate of Pneumocystis colonization among HIV-infected patients at autopsy and analyze associated clinical variables. METHODS: Subjects were selected from the Multicenter AIDS Cohort Study. Subjects who died from causes other than Pneumocystis pneumonia and consented to autopsy were included in analysis. DNA was extracted from lung tissue, and nested PCR was performed to detect the presence of Pneumocystis. Clinical data were obtained from the Multicenter AIDS Cohort database. Univariate and multivariate analyses were performed to determine predictors of Pneumocystis colonization. RESULTS: Pneumocystis DNA was detected in 42 of 91 (46%) subjects by nested PCR. Clinical variables such as CD4 cell count, use of Pneumocystis prophylaxis or antiretroviral drugs, and history of previous Pneumocystis pneumonia were not related to risk of colonization. Multivariate analysis demonstrated that cigarette smoking was related to an increased risk of colonization [odds ratio (OR), 4.5; 95% confidence interval (CI), 1.27-15.6; P = 0.02] and risk also varied by city of residence (OR, 0.12; 95% CI, 0.03-0.45; P = 0.002 for living in Los Angeles). CONCLUSIONS: This study found a high rate of Pneumocystis colonization among HIV-infected patients. We also identified cigarette smoking and city of residence as novel, independent risk factors for colonization. The role of subclinical colonization in disease transmission and the effects of Pneumocystis colonization on the lung require further study.