Mouse Keratinocytes Without Keratin Intermediate Filaments Demonstrate Substrate Stiffness Dependent Behaviors

Area of Science:

• Cell Biology
• Biophysics
• Biochemistry

Background:

• Actin microfilaments and keratin intermediate filaments (IFs) were traditionally viewed as having distinct mechanical roles.
• Emerging evidence suggests functional interdependence between these cytoskeletal networks and cell adhesion structures.
• Keratin IFs are crucial for epithelial cell structure and function.

Purpose of the Study:

• To investigate the impact of keratin IF loss on the colony formation kinematics of mouse keratinocytes.
• To determine if keratin IFs influence cell behavior and mechanical responses on substrates of varying stiffness.
• To explore the interdependence between actin and keratin networks during epithelial sheet formation.

Main Methods:

• Time-lapse imaging and deformation tracking microscopy were employed to monitor colony formation in wild-type (WT) and keratin-deficient (KO) mouse keratinocytes over 24 hours.
• Cells were cultured on soft (1.2 kPa) and stiff (24 kPa) collagen-coated substrates under high calcium conditions.
• Immunofluorescence staining was used to analyze the organization of actin and key adhesion proteins.

Main Results:

• Keratin deficiency significantly altered cell morphology, spread area, and the organization of cytoskeletal and adhesion proteins on both soft and stiff substrates.
• Absence of keratin IFs markedly reduced the capacity of keratinocytes to mechanically deform soft substrates.
• Keratin-deficient cells exhibited more efficient colony formation on stiff substrates compared to soft ones, the inverse of WT cell behavior.

Conclusions:

• The findings strongly support an interdependence between actin microfilaments and keratin IFs.
• Keratin IFs play a significant role in keratinocyte mechanosensation, influencing their response to substrate stiffness.
• Keratin IFs are an important, potentially under-recognized, component of the force generation apparatus in epithelial cells.