7th Week of Research

Hi Readers,

During my 7th week of research, I began working with a mutant strain of C. elegans that does not express the anaphase promoting complex. The APC mutant emb-27 will arrest cells in metaphase I of meiosis, but other cellular processes will continue to occur after anaphase I should be occurring; for example, the residual body still forms when spermatids should be budding from the residual body. This mutant will allow us to continue to characterize SUMO’s role throughout the meiotic division zone, even when the chromatin is not moving through the sequential cellular divisions. I have had some hiccups with the permeabilization step required to allow the SUMO antibody access to SUMO, which is within the cells of the gonad. I will try varying amounts of permeabilization in my next prep to determine the sufficient amount to allow visualization of SUMO.

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6th Week of Summer Research

Hi Readers,

In our cellular and developmental biology lab, we utilize different fixations to visualize the chromatin and proteins of interest. The two major fixations we utilize are an overnight methanol and 10 – 15 minute paraformaldehyde (aldehyde). Methanol fixations precipitate the proteins and preserve the structure of the proteins, but can cause denaturing of small soluble molecules. Paraformaldehyde cross links proteins by free amine groups, which ultimately forms intermolecular bridges (1).

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5th Week of Summer Research

Good Afternoon Readers,

Throughout previous SUMO preps, I have noticed an irregular, punctate SUMO pattern in a cellular stage called diplotene. The punctuates seemed to be confined within the nuclear envelope, but I wanted to test this hypothesis. To test my hypothesis, I began investigating the pattern of SUMO and a nuclear envelope (NE) protein, LMN-1. I utilized a co-staining procedure with SUMO and LMN-1, but both of these proteins require the same secondary antibody, which means they will both fluoresce the same color: to mitigate this challenge, I used controls for both LMN-1 and SUMO to show their independent patterns, in addition to co-stained slides with SUMO and LMN-1. Upon analysis of the LMN-1 controls, the staining was showing LMN-1 associating with the chromatin, but not staining the nuclear envelope. This is an uncharacteristic pattern for LMN-1: because LMN-1 is a nuclear envelope protein, the staining should be on the NE, but not the chromatin. Due to the irregular pattern of LMN-1, I could not draw any conclusions from this SUMO/LMN-1 prep.

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4th Week of Research

Good Afternoon Readers,

In my fourth week of research, I noticed an interesting progression of SUMO during the karyosome stage. One can see cells progressing from one stage to the next; for example, cells will be captured moving from the diplotene stage to the karyosome stage (i.e. the cells will exhibit increasing chromatin compaction). I observed a correlation between the chromatin becoming more compacted as it becomes the characteristic fully-compacted stage of karyosome with SUMO moving from the outer edge of the chromatin to completely associating with fully-compacted chromatin. This is an interesting development that will allow further investigation into the role of SUMO and chromatin compaction.

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3rd Week of Research

Good Afternoon Readers,

In my third week of research in the Shakes’ lab, I worked with a fellow student researcher in the Kerscher lab to utilize their UTAG protocol on my nematode gonads. The kmUTAG protocol is developed from one of the SUMO proteases, Ulp-1; further, kmUTAG is a useful tool of evaluation because it has less affinity to bind to free, unconjugated SUMO. A quick note about the SUMOylation pathway, three enzymes are required for a protein to be SUMOylated. The three enzymes are known as the activating enzyme – E1, the conjugating enzyme – E2, and the ligase E3 – in nematodes, the enzymes frequently studied for their localization throughout nematode gonads, especially during the meiotic divisions are the conjugating enzyme (E2) UBC-9 and the ligase (E3) GEI-17. I have included a pathway depiction developed by Dr. Shakes at the bottom of this post.

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