Protein kinases direct polarized growth by regulating the cytoskeleton in time and space and could play similar roles in cell division. We found that the Cdc42-activated polarity kinase Pak1 colocalizes with the assembling contractile actomyosin ring (CAR) and remains at the division site during septation. Mutations in pak1 led to defects in CAR assembly and genetic interactions with cytokinesis mutants. Through a phosphoproteomic screen, we identified novel Pak1 substrates that function in polarized growth and cytokinesis. For cytokinesis, we found that Pak1 regulates the localization of its substrates Mid1 and Cdc15 to the CAR. Mechanistically, Pak1 phosphorylates the Mid1 N-terminus to promote its association with cortical nodes that act as CAR precursors. Defects in Pak1-Mid1 signaling lead to misplaced and defective division planes, but these phenotypes can be rescued by synthetic tethering of Mid1 to cortical nodes. Our work defines a new signaling mechanism driven by a cell polarity kinase that promotes CAR assembly in the correct time and place.

The lack of standardization of tissue handling and processing hinders the development and validation of new biomarkers in research and clinical settings. We compared the histomorphology and the quality and quantity of biomolecules in paraffin-embedded mouse tissues, followed by fixation with neutral buffered formalin (NBF), 70% ethanol, and buffered ethanol (BE70) fixative. The quality of the histomorphology and immunohistochemistry in BE70 was relatively time-independent, whereas those in NBF rapidly decreased after 1 week of fixation. Protein recovered from tissue fixed in 70% ethanol and BE70 was compatible with Western blot and protein array using AKT and GAPDH antibodies, regardless of the fixation time. In addition, the quality and quantity of RNA extracted from tissue in ethanol-based fixative showed minimal changes from 4 hr to 6 months, whereas NBF had a dramatic detrimental change in RNA quality after 1 week of fixation. Furthermore, ethanol-based fixative offers a superior DNA template for PCR amplification-based molecular assays than NBF. In conclusion, coagulative, ethanol-based fixatives show a broader time spectrum than the aldehyde crosslinking fixative NBF in their histomorphological features and the quantity and quality of the biomolecules from paraffin-embedded tissue, and they may facilitate the use of fixative-fixed paraffin-embedded tissues in research and clinical laboratories, avoiding overfixation.

SummaryCD8 T‐cells are an essential component of the adaptive immune response accountable for the clearance of virus‐infected cells via cytotoxic effector functions. Maintaining a specific metabolic profile is necessary for these T‐cells to sustain their effector functions and clear pathogens. When CD8 T‐cells are activated via T‐cell receptor recognition of viral antigen, they transition from a naïve to an effector state and eventually to a memory phenotype, and their metabolic profiles shift as the cells differentiate to accomidate different metabolic demands. However, in the context of particular chronic viral infections (CVIs), CD8 T‐cells can become metabolically dysfunctional in a state known as T‐cell exhaustion. In this state, CD8 T‐cells exhibit reduced effector functions and are unable to properly control pathogens. Clearing these chronic infections becomes progressively difficult as increasing numbers of the effector T‐cells become exhausted. Hence, reversal of this dysfunctional metabolic phenotype is vital when considering potential treatments of these infections and offers the opportunity for novel strategies for the development of therapies against CVIs. In this review we explore research implicating alteration of the metabolic state as a means to reverse CD8 T‐cell exhaustion in CVIs. These findings indicate that strategies targeting dysfunctional CD8 T‐cell metabolism could prove to be a promising option for successfully treating CVIs.